Signaling pathways underlying the antidepressant-like effect of inosine in mice

Abstract

Inosine is a purine nucleoside formed by the breakdown of adenosine that elicits an antidepressant-like effect in mice through activation of adenosine A₁ and A_2A receptors. However, the signaling pathways underlying this effect are largely unknown. To address this issue, the present study investigated the influence of extracellular-regulated protein kinase (ERK)1/2, Ca²⁺/calmoduline-dependent protein kinase (CaMKII), protein kinase A (PKA), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase 3beta (GSK-3β) modulation in the antiimmobility effect of inosine in the tail suspension test (TST) in mice. In addition, we attempted to verify if inosine treatment was capable of altering the immunocontent and phosphorylation of the transcription factor cyclic adenosine monophosphatate (cAMP) response-binding element protein (CREB) in mouse prefrontal cortex and hippocampus. Intracerebroventricular administration of U0126 (5 μg/mouse, MEK1/2 inhibitor), KN-62 (1 μg/mouse, CaMKII inhibitor), H-89 (1 μg/mouse, PKA inhibitor), and wortmannin (0.1 μg/mouse, PI3K inhibitor) prevented the antiimmobility effect of inosine (10 mg/kg, intraperitoneal (i.p.)) in the TST. Also, administration of a sub-effective dose of inosine (0.1 mg/kg, i.p.) in combination with a sub-effective dose of AR-A014418 (0.001 μg/mouse, GSK-3β inhibitor) induced a synergic antidepressant-like effect. None of the treatments altered locomotor activity of mice. Moreover, 24 h after a single administration of inosine (10 mg/kg, i.p.), CREB phosphorylation was increased in the hippocampus. Our findings provided new evidence that the antidepressant-like effect of inosine in the TST involves the activation of PKA, PI3K/Akt, ERK1/2, and CaMKII and the inhibition of GSK-3β. These results contribute to the comprehension of the mechanisms underlying the purinergic system modulation and indicate the intracellular signaling pathways involved in the antidepressant-like effect of inosine in a preclinical test of depression.

Keywords: CREB; Depression; Inosine; Protein kinases; Signaling pathways.

Fig. 1

Involvement of MEK/ERK1/2 pathway in the antidepressant-like effect of inosine in the TST. Effect of treatment of mice with the MEK1/2 inhibitor U0126 (5 μg/mouse, i.c.v.) on the antidepressant-like effect of inosine (10 mg/kg, i.p.) in the TST (a) and locomotor activity in the OFT (b). Values are expressed as SEM of six to eight mice. **p < 0.01 compared with the vehicle-treated control group. ^## p < 0.01 compared with inosine-treated group

Fig. 2

Involvement of CaMKII in the antidepressant-like effect of inosine in the TST. Effect of treatment of mice with the CaMKII inhibitor KN-62 (1 μg/mouse, i.c.v.) on the antidepressant-like effect of inosine (10 mg/kg, i.p.) in the TST (a) and locomotor activity in the OFT (b). Values are expressed as SEM of six to eight mice. ***p < 0.001 compared with the vehicle-treated control group. ^### p < 0.001 compared with inosine-treated group

Fig. 3

Involvement of PKA in the antidepressant-like effect of inosine in the TST. Effect of treatment of mice with the PKA inhibitor H-89 (1 μg/mouse, i.c.v.) on the inosine-induced (10 mg/kg, i.p.) antidepressant-like effect in the TST (a) and locomotor activity in the OFT (b). Values are expressed as SEM of seven to eight mice. *p < 0.05 compared with the vehicle-treated control group. ^# p < 0.05 compared with inosine-treated group

Fig. 4

Involvement of PI3K/Akt in the antidepressant-like effect of inosine in the TST. Effect of treatment of mice with the PI3K inhibitor wortmannin (0.1 μg/mouse, i.c.v.) on the inosine-induced (10 mg/kg, i.p.) antidepressant-like effect in the TST (a) and locomotor activity in the OFT (b). Values are expressed as mean + SEM of six to eight mice. **p < 0.01 compared with the vehicle-treated control group. ^## p < 0.01 compared with inosine-treated group

Fig. 5

Involvement of GSK-3β inhibition in the antidepressant-like effect of inosine in the TST. Effect of treatment of mice with the GSK-3β inhibitor AR-A014418 (0.001 mg/mouse, i.c.v.) in combination with a sub-effective dose of inosine (0.1 mg/kg, p.m.) in the TST (a) and locomotor activity in the OFT (b). Values are expressed as mean ± SEM of seven mice. **p < 0.01 compared with the vehicle-treated control group

Fig. 6

Effect of inosine treatment (10 mg/kg, i.p.) on CREB immunocontent and phosphorylation (Ser133) in the hippocampus and prefrontal cortex of mice. a, b The representative western blotting and the quantification of phospho-CREB (P-CREB) and total immunocontent of CREB (T-CREB) in the hippocampus (a) and prefrontal cortex of mice (b) 30 min after inosine administration. c, d The representative western blotting and the quantification of P-CREB and T-CREB in hippocampus (c) and prefrontal cortex of mice (d) 24 h after inosine administration. Results were quantified as percent of control and expressed as mean ± SEM of seven to eight mice. *p < 0.05 compared with the vehicle-treated control group