STIMULATION OF THE HYPOTHALAMO-PITUITARY-ADRENAL AXIS IN THE RAT BY 3SELECTIVE TYPE-4 PHOSPHODIESTERASE INHIBITORS - IN-VITRO AND IN-VIVO STUDIES

1 Previous studies in our laboratory have shown that the synthetic xan thine analogue denbufylline, a selective type 4 phosphodiesterase (PDE -4) inhibitor, is a potent activator of the hypothalamo-pituitary-adre nal (HPA) axis when given orally or intraperitoneally (i.p.) to adult male rats. This paper describes the results of experiments in which we ll established in vivo and in vitro methods were used to compare the e ffects of denbufylline on HPA function with those of two other selecti ve PDE-4 inhibitors, rolipram and BRL 61063 (1,3-dicyclopropylmethyl-8 -amino-xanthin For comparison, parallel measurements of the immunoreac tive- (ir-) luteinising hormone (LH) were made where appropriate. 2 Wh en injected intraperitoneally, rolipram (40 and 200 mu g kg(-1), P < 0 .005), denbufylline (0.07-0.6 mu g kg(-1), P < 0.05) and BRL 61063 (30 mu g kg(-1), P < 0.005) each produced marked rises in the serum ir-co rticosterone concentrations. However, lower doses of rolipram (1.6 and 8 mu g kg(-1)) and BRL 61063 (0.25-6 mu g kg(-1)) were without effect (P > 0.05). By contrast, intracerebroventricular (i.c.v.) injection o f rolipram (8 ng-1 mu g kg(-1)) or denbufylline (50 ng-1 mu g kg(-1)) failed to influence the serum ir-corticosterone concentration. BRL 610 63 (8-120 ng kg(-1), i.c.v.) was also ineffective in this regard altho ugh at a higher dose (1 mu g kg(-1), i.c.v.) it produced a small but s ignificant (P < 0.05) increase in ir-corticosterone release. Denbufyll ine also increased the serum ir-LH concentration when given peripheral ly (0.2-0.6 mu g kg(-1), i.p., P < 0.05) or centrally (100 ng kg(-1), i.c.v., P < 0.05) but rolipram (1.6-200 mu g kg(-1), i.p. or 8 ng(-1) mu g kg(-1), i.c.v.) and BRL 61063 (0.25-30 mu g kg(-1), i.p. or 1 ng- 1 mu g kg(-1), i.c.v.) did not (P > 0.05). 3 In vitro rolipram (10 mu M, P < 0.01), denbufylline (1 mM, P < 0.001) and BRL 61063 (1 and 10 m u M, P < 0.05) stimulated the release of corticotrophin releasing horm one (ir-CRH-41) but lower concentrations of the drugs were without eff ect as also was BRL 61063 at 100 mu M (P > 0.05); the rank order of po tency was thus BRL 61063 > rolipram > denbufylline. The adenylyl cycla se activator forskolin (100 mu M, P < 0.01) also stimulated the releas e of ir-CRH-41, producing effects which were additive with those of ro lipram and denbufylline but not with those of BRL 61063. The secretory responses to forskolin (100 mu M) were accompanied by a highly signif icant increase in the cyclic AMP content of the hypothalamic tissue (P < 0.005). Rolipram (10 mu M) also significantly (P < 0.05) elevated t he hypothalamic cyclic AMP but denbufylline (10 mM) and BRL 61063 (10 mu M) did not. However, all three PDE-inhibitors potentiated the rise in cyclic AMP induced by forskolin (P < 0.05). None of the drugs teste d, alone or in combination, modified the release of arginine vasopress in (ir-AVP) from the hypothalamus. 4 Rolipram (100 mu M), denbufylline (100 mu M) and BRL 61063 (100 mu M) stimulated the release of cortico trophin (ir-ACTH) from pituitary tissue in vitro (P < 0.05) but in low er concentrations they were without significant effect. In addition, r olipram (10 mu M, P < 0.05), denbufylline (0.1 mu M, P < 0.05) and BRL 61063 (10 mu M, P < 0.05) potentiated the significant (P < 0.05) rise s in ir-ACTH secretion induced by forskolin (100 mu M). Forskolin (100 mu M) also produced a highly significant increase (P < 0.01) in the t issue cyclic AMP content which was further potentiated by rolipram (10 mu M), denbufylline (10 mu M) and BRL 61063 (10 mu M) which, alone di d not affect the cyclic AMP content of the tissue. 5 Since both denbuf ylline and BRL 61063 possess significant adenosine A(1) receptor block ing activity, further studies examined the potential influence of thes e receptors on the secretion in vitro of CRH-41, AVP and ACTH. The rel ease of ir-CRH-41 was increased significantly by adenosine deaminase ( ADA, 5 u ml(-1), P < 0.05) and the A(1)-receptor antagonist, 1,3-dicyc lopropyl-8-cyclopentylxanthine (DPCPX, 0.1-10 nM, P < 0.05). The respo nses to ADA were abolished by the A(1) receptor agonist N-6-cyclohexyl adenosine (CHA, 100 nM, P < 0.05) which alone had no significant effec t on ir-CRH-41 release. ADA (0.1-10 u ml(-1)) and DPCPX (1 nM) had wea k stimulant and inhibitory effects, respectively, on the release of ir -ACTH from the pituitary gland while CHA (0.1-10 nM) was without effec t. Ligand binding studies with [H-3]-DPCPX as a probe demonstrated the presence of specific high affinity A(1) binding sites in the hypothal amus (K-d=0.7 nM; B-max=367+/-32 fmol mg(-1) protein) and in the hippo campus (K-d=1 nM; B-max=1165+/-145 fmol mg(-1) protein). In both tissu es binding of the ligand was displaced by CHA (IC50=1 nM (hypothalamus ) and 2 nM (hippocampus)), BRL 61063 (IC50=80 nM (hypothalamus) and 10 0 nM (hippocampus)) and denbufylline (IC50=5 mu M (hypothalamus) and 9 mu M (hippocampus)) but not by rolipram. 6 The results suggest that r olipram, denblufylline and BRL 61063 stimulate the HPA axis in the rat , acting at the levels of both the hypothalamus and the pituitary glan d. Their actions may be explained, at least in part, by inhibition of PDE-4 but additional actions including blockade of hypothalamic adenos ine Al receptors by denbufylline and BRL 61063 cannot be excluded.