adp-beta-s and 1-3-dipropyl-8-cyclopentylxanthine

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effect

Topics: Adenosine Deaminase; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cell Line; Estrenes; Female; Glucose; Insulin; Insulinoma; Islets of Langerhans; Male; Pancreatic Neoplasms; Phosphodiesterase Inhibitors; Purinergic Agonists; Purinergic Antagonists; Purines; Pyrrolidinones; Rats; Rats, Wistar; Thionucleotides; Uridine Triphosphate; Xanthines

Experiments on hippocampal slices were carried out in order to find out whether the release of noradrenaline in the hippocampus can be modulated through P2-receptors. The slices were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM), and stimulated electrically, in most experiments by 4 pulses/100 Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA) and the nucleotides ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) decreased the evoked overflow of tritium by up to 55%. The adenosine A2a-agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamido-adenosin e (CGS 21680; 0.003-0.3 microM) caused no change. The concentration-response curve of CPA was shifted to the right by the A1-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3 nM) but not by the P2-receptor antagonists cibacron blue 3GA (30 microM) and reactive blue 2 (30 microM); the apparent pKB value of DPCPX against CPA was 9.0. In contrast, the concentration-response curve of ATP was shifted to the right by DPCPX (3 nM), apparent pKB 8.7, as well as by cibacron blue 3GA (30 microM), apparent pKB 5.2, and reactive blue 2 (30 microM), apparent pKB 5.6; the antagonist effects of DPCPX and cibacron blue 3GA were additive in a manner compatible with the blockade of two separate receptors for ATP. The same pattern was obtained with ATP gamma S: its concentration-response curve was shifted to the right by DPCPX as well as by cibacron blue 3GA and reactive blue 2. Suramin (300 microM) antagonized neither the effect of ATP nor that of ATP gamma S. The 5'-nucleotidase inhibitor alpha, beta-methylene-ADP (100 microM) did not change the effect of ATP. Only cibacron blue 3GA (30 microM) but not reactive blue 2 (30 microM), given alone, consistently caused a small increase of the evoked overflow of tritium. Hippocampal slices degraded exogenous ATP, and this degradation was reduced by cibacron blue 3GA (30 microM), reactive blue 2 (30 microM) and suramin (300 microM). The results indicate that the noradrenergic terminal axons of the rat hippocampus possess P2-receptors in addition to the known A1-adenosine receptors. The presynaptic P2-receptors mediate an inhibition of noradrenaline release, are activated by nucleotides but not nucleosides, and are blocked by cibacron blue 3GA and reactive blue 2. ATP and ATP gamma S act at both the A1- and the P2-receptors. An autoreceptor function

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Electric Stimulation; Hippocampus; In Vitro Techniques; Isotope Labeling; Male; Norepinephrine; Phenethylamines; Rats; Rats, Wistar; Receptors, Purinergic P2; Thionucleotides; Triazines; Tritium; Xanthines; Yohimbine

The classification of purinergic receptors is seriously hampered by the lack of specific antagonists. Furthermore, there is increasing evidence that other purinoceptor subtypes may exist that are different than the relatively well characterized P2X, P2Y, P2Z and P2T. Human isolated urinary bladder was reported to contract in response to challenge with alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) and adenosine 5'-triphosphate (ATP), probably through activation of P2X purinoceptors. In this work, we tried to classify the purinoceptors subtypes present in human detrusor muscle by using adenosine 5'-[beta-thio]diphosphate (ADP beta S), alpha,beta-MeATP, 2-methylthio adenosine 5'-triphosphate (2-MeSATP), ATP and uridine 5'-triphosphate (UTP). We also examined the activity of two putative P2 antagonists (p-chloromercuribenzensulfonic acid [PCMBS] and Reactive Blue 2 [RB-2]). The agonist rank order of potency was alpha,beta-MeATP = ADP beta S > 2-MeSATP > ATP >> UTP. Cumulative responses to alpha,beta-MeATP induced a very rapid desensitization, but responses to alpha,beta-MeATP and ADP beta S, both at 100 microM, were additive. PCMBS antagonized ADP beta S-induced contractions with a pKB of 6.49, but it was inactive against alpha,beta-MeATP. The putative P2Y antagonist RB-2 had no effect against ADP beta S-induced contraction. We conclude that human detrusor muscle contains two contractile purinoceptor subtypes. One is activated by alpha,beta-MeATP and is probably the P2X subtype; the other is activated by ADP beta S and appears to be different from those accepted by the current classification. The similarity between our results and those obtained by other investigators is discussed.

Topics: 4-Chloromercuribenzenesulfonate; Adenosine Diphosphate; Adenosine Triphosphate; Humans; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Receptors, Purinergic P2; Thionucleotides; Triazines; Urinary Bladder; Xanthines