Synthetic, nondegradable diadenosine polyphosphates and diinosine polyphosphates: their effects on insulin-secreting cells and cultured vascular smooth muscle cells.
Diadenosine polyphosphates show a dissimilarity between their effects in static and perifusion experiments with respect to insulin release that may be due to degradation of the compounds. The aim was to investigate two nondegradable compounds of bisphosphorothioates containing a methylene or chloromethylene group (namely, diadenosine 5',5' "-(P(1),P(4)-dithio-P(2),P(3)-methylene)tetraphosphate and diadenosine 5',5' "-(P(1),P(4)-dithio-P(2),P(3)-chloromethylene)tetraphosphate), as mixtures of three or four diastereomers. Owing to their modified structures, these compounds are resistant to degradation (ectophosphodiesterases, diphosphohydrolases, and phosphorylases). Both compounds tested were minimally degraded (2%) even after 16 h when incubated with insulin-secreting (INS-1) cells. Additionally, diinosine polyphosphates (Ip(5)I and Ip(6)I), putative antagonists of diadenosine polyphosphates, were tested. By use of [(3)H]Ap(4)A, saturable binding sites for both diadenosine polyphosphate analogues were found in INS-1 cells, 3T3 preadipocyte cells, and vascular smooth muscle cells (VSMC) and for both Ip(5)I and Ip(6)I in INS-1 cells. The synthesized diadenosine polyphosphate analogues have the same affinity as Ap(4)A, whereas Ip(5)I and Ip(6)I inhibit binding at higher concentrations (10-100 microM). Insulin release was investigated in static experiments over 90 min in INS-1 cells. Insulin release was inhibited dose-dependently by both of the diadenosine polyphosphate analogues to the same degree as by Ap(4)A. The glucose-induced insulin release curve was not shifted to the right. Both compounds inhibit insulin release only at high (insulin stimulatory) glucose concentrations, e.g., 5.6 mM glucose. Ip(5)I and Ip(6)I antagonized Ap(5)A-mediated inhibition of insulin release. [(3)H]Thymidine incorporation into VSMC was not influenced by either synthetic diadenosine polyphosphate analogue, indicating that Ap(4)A does not act by itself in this case but (active) degradation products mediate the effect. The data indicate the following. (1) Since nondegradable compounds inhibit insulin release as well as Ap(4)A, it is Ap(4)A itself and not any of its degradation products that induces this effect. (2) Diadenosine polyphosphate effects on cell proliferation are mediated via a degradation product in contrast to their effect on insulin release. (3) Ip(5)I and Ip(6)I act like antagonists. Both synthetic analogues and diinosine polyphosphates are valuable tools for diabetes research.
Verspohl
Eugen J
EJ
Department of Pharmacology, Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, Germany. verspoh@uni-muenster.de
Blackburn
G Michael
GM
Hohmeier
Nina
N
Hagemann
Joost
J
Lempka
Matthias
M
D016475:3T3 Cells; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D015226:Dinucleoside Phosphates / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D011485:Protein Binding; D051381:Rats; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013936:Thymidine / Q000378:metabolism
D015226:Dinucleoside Phosphates; D007328:Insulin; C473681:diadenosine 5',5'''-(P(1),P(4)-dithio-P(2),P(3)-chloromethylene)tetraphosphate; C473680:diadenosine 5',5'''-(P(1),P(4)-dithio-P(2),P(3)-methylene)tetraphosphate; D013936:Thymidine