Daily cycling of nitric oxide synthase (NOS) in the hippocampus of pigeons (C. livia)

Abstract

Background: Nitric oxide synthase (NOS) is essential for the synthesis of nitric oxide (NO), a non-conventional neurotransmitter with an important role in synaptic plasticity underlying processes of hippocampus-dependent memory and in the regulation of biological clocks and circadian rhythms. Many studies have shown that both the NOS cytosolic protein content and its enzymatic activity present a circadian variation in different regions of the rodent brain, including the hippocampus. The present study investigated the daily variation of NOS enzymatic activity and the cytosolic content of nNOS in the hippocampus of pigeons.

Results: Adult pigeons kept under a skeleton photoperiod were assigned to six different groups. Homogenates of the hippocampus obtained at six different times-of-day were used for NOS analyses. Both iNOS activity and nNOS cytosolic protein concentrations were highest during the subjective light phase and lowest in the subjective dark phase of the circadian period. ANOVA showed significant time differences for iNOS enzymatic activity (p < 0.05) and for nNOS protein content (p < 0.05) in the hippocampus. A significant daily rhythm for both iNOS and nNOS was confirmed by analysis with the Cosinor method (p < 0.05). The present findings indicate that the enzymatic activity of iNOS and content of nNOS protein in the hippocampus of pigeons exhibit a daily rhythm, with acrophase values occurring during the behavioral activity phase.

Conclusions: The data corroborate the reports on circadian variation of NOS in the mammalian hippocampus and can be considered indicative of a dynamic interaction between hippocampus-dependent processes and circadian clock mechanisms.

Figure 1

Enzymatic activity of cNOS and iNOS in hippocampus of pigeon. A- Mean values (+ s.e.m.) of the amount of L-citrulline (pmol/min/mg) produced by the reaction of the enzymatic activity of Ca²⁺ - dependent NOS (cNOS) in samples of hippocampal tissue homogenate. Data are expressed as ratio calculated relative to the mean of ZT0 group. One-way ANOVA. B- Mean values (+ s.e.m.) in the amount of L-citrulline (pmol/min/mg) produced by the reaction of the enzymatic activity of Ca²⁺ - independent NOS (iNOS) in samples of hippocampal tissue homogenate expressed as ratio relative to the mean of ZT0 group. One-way ANOVA indicated between group differences (p < 0.001); * indicates p < 0.05 when compared to ZT8, ZT12 and ZT16; # indicates p < 0.05 compared with ZT12 and (+ s.e.m.) ZT16 (Tukey-Kramer test; n = 4 pigeons per group).

Figure 2

Protein expression of nNOS in hippocampus of pigeon. A- Mean values (+ s.e.m.) of optical densitometric analyses of immunoreactive bands for nNOS in samples of hippocampal tissue homogenate normalized with Ponceau staining. Each mean was calculated with reference to the ZT0 value. The dashed line indicates the fitted curve by the Cosinor method. One-way ANOVA indicated group effects (p < 0.001); * indicates p < 0.05 when compared to ZT12, ZT16 and ZT20; # indicates p < 0.05 compared with ZT16 and ZT20 (Turkey-Kramer test; n = 4 pigeons per group). B- Representative immunoreactive bands for nNOS and proteins detected by Ponceau staining in each group.