Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by three selective type-4 phosphodiesterase inhibitors: in vitro and in vivo studies

Abstract

1. Previous studies in our laboratory have shown that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE-4) inhibitor, is a potent activator of the hypothalamo-pituitary-adrenal (HPA) axis when given orally or intraperitoneally (i.p.) to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro methods were used to compare the effects of denbufylline on HPA function with those of two other selective PDE-4 inhibitors, rolipram and BRL 61063 (1,3-dicyclopropylmethyl-8-amino-xanthine). For comparison, parallel measurements of the immunoreactive- (ir-) luteinising hormone (LH) were made where appropriate.

2. When injected intraperitoneally, rolipram (40 and 200 μg kg⁻¹, P<0.005), denbufylline (0.07–0.6 μg kg⁻¹, P<0.05) and BRL 61063 (30 μg kg⁻¹, P<0.005) each produced marked rises in the serum ir-corticosterone concentrations. However, lower doses of rolipram (1.6 and 8 μg kg⁻¹) and BRL 61063 (0.25–6 μg kg⁻¹) were without effect (P>0.05). By contrast, intracerebroventricular (i.c.v.) injection of rolipram (8 ng–1 μg kg⁻¹) or denbufylline (50 ng–1 μg kg⁻¹) failed to influence the serum ir-corticosterone concentration. BRL 61063 (8–120 ng kg⁻¹, i.c.v.) was also ineffective in this regard although at a higher dose (1 μg kg⁻¹, i.c.v.) it produced a small but significant (P<0.05) increase in ir-corticosterone release. Denbufylline also increased the serum ir-LH concentration when given peripherally (0.2–0.6 μg kg⁻¹, i.p., P<0.05) or centrally (100 ng kg⁻¹, i.c.v., P<0.05) but rolipram (1.6–200 μg kg⁻¹, i.p. or 8 ng–1 μg kg⁻¹, i.c.v.) and BRL 61063 (0.25–30 μg kg⁻¹, i.p. or 1 ng–1 μg kg⁻¹, i.c.v.) did not (P>0.05).

3. In vitro rolipram (10 μM, P<0.01), denbufylline (1 mM, P<0.001) and BRL 61063 (1 and 10 μM, P<0.05) stimulated the release of corticotrophin releasing hormone (ir-CRH-41) but lower concentrations of the drugs were without effect as also was BRL 61063 at 100 μM (P>0.05); the rank order of potency was thus BRL 61063>rolipram>denbufylline. The adenylyl cyclase activator forskolin (100 μM, P<0.01) also stimulated the release of ir-CRH-41, producing effects which were additive with those of rolipram and denbufylline but not with those of BRL 61063. The secretory responses to forskolin (100 μM) were accompanied by a highly significant increase in the cyclic AMP content of the hypothalamic tissue (P<0.005). Rolipram (10 μM) also significantly (P<0.05) elevated the hypothalamic cyclic AMP but denbufylline (10 mM) and BRL 61063 (10 μM) did not. However, all three PDE-inhibitors potentiated the rise in cyclic AMP induced by forskolin (P<0.05). None of the drugs tested, alone or in combination, modified the release of arginine vasopressin (ir-AVP) from the hypothalamus.

4. Rolipram (100 μM), denbufylline (100 μM) and BRL 61063 (100 μM) stimulated the release of corticotrophin (ir-ACTH) from pituitary tissue in vitro (P<0.05) but in lower concentrations they were without significant effect. In addition, rolipram (10 μM, P<0.05), denbufylline (0.1 μM, P<0.05) and BRL 61063 (10 μM, P<0.05) potentiated the significant (P<0.05) rises in ir-ACTH secretion induced by forskolin (100 μM). Forskolin (100 μM) also produced a highly significant increase (P<0.01) in the tissue cyclic AMP content which was further potentiated by rolipram (10 μM), denbufylline (10 μM) and BRL 61063 (10 μM) which, alone did not affect the cyclic AMP content of the tissue.

5. Since both denbufylline and BRL 61063 possess significant adenosine A₁ receptor blocking activity, further studies examined the potential influence of these receptors on the secretion in vitro of CRH-41, AVP and ACTH. The release of ir-CRH-41 was increased significantly by adenosine deaminase (ADA, 5 u ml⁻¹, P<0.05) and the A₁-receptor antagonist, 1,3-dicyclopropyl-8-cyclopentylxanthine (DPCPX, 0.1–10 nM, P<0.05). The responses to ADA were abolished by the A₁ receptor agonist N⁶-cyclo-hexyladenosine (CHA, 100 nM, P<0.05) which alone had no significant effect on ir-CRH-41 release. ADA (0.1–10 u ml⁻¹) and DPCPX (1 nM) had weak stimulant and inhibitory effects, respectively, on the release of ir-ACTH from the pituitary gland while CHA (0.1–10 nM) was without effect. Ligand binding studies with [³H]-DPCPX as a probe demonstrated the presence of specific high affinity A₁ binding sites in the hypothalamus (K_d=0.7 nM; B_max=367±32 fmol mg⁻¹ protein) and in the hippocampus (K_d=1 nM; B_max=1165±145 fmol mg⁻¹ protein). In both tissues binding of the ligand was displaced by CHA (IC₅₀=1 nM (hypothalamus) and 2 nM (hippocampus)), BRL 61063 (IC₅₀=80 nM (hypothalamus) and 100 nM (hippocampus)) and denbufylline (IC₅₀=5 μM (hypothalamus) and 9 μM (hippocampus)) but not by rolipram.

6. The results suggest that rolipram, denblufylline and BRL 61063 stimulate the HPA axis in the rat, acting at the levels of both the hypothalamus and the pituitary gland. Their actions may be explained, at least in part, by inhibition of PDE-4 but additional actions including blockade of hypothalamic adenosine A₁ receptors by denbufylline and BRL 61063 cannot be excluded.

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