CARBON MONOXIDE-MEDIATED HUMORAL PATHWAY
FOR THE TRANSMISSION OF LIGHT SIGNAL TO THE HYPOTHALAMUS

Carbon monoxide (CO), a product of heme degradation by heme oxygenase (HO), is known as a gaseous paracrine regulatory factor produced exclusively for local regulation. There is, however, evidence that it is not involved solely in local regulation (1, 2). The expression of the HO system and the ability to produce CO have been demonstrated in animal organs, including the eye (3-6). In addition, the induction of HO-1 by intense visible light was demonstrated in the retina in rats (7). Recently, it was discovered that CO was released from the eye into the outflowing venous blood in males of a wild boar and domestic pig crossbreed in a manner dependent on ambient light (8).

Ophthalmic venous blood (OphVB) flows from the ophthalmic sinus (OphS) to the venous cavernous sinus (VCS) of the perihypophyseal vascular complex (PVC), located at the base of the brain (9). The PVC is formed from the VCS, filled with a veno-venous network, and the internal carotid artery (ICA) or its rete mirabile passing the VCS (10). The distance between the streams of arterial and venous blood is greatly reduced in this structure. The permeation of several regulatory factors including dopamine, β-endorphin, GnRH, oxytocin, and steroid male pheromone (molecular weight of 0.19–3.4 kDa) from venous blood to arterial blood has been shown in the PVC in the rabbit, sheep, and pig (11-18). We hypothesised that molecules of CO (28.8 Da) transported with blood filling the venous network of the VCS can penetrate the walls of blood vessels and permeate to the arterial blood of the ICA supplying the brain. The theoretical model of humoral phototransduction presented by Oren (19) proposed that countercurrent exchange in the VCS of CO produced in the retina could allow direct access of the gaseous messenger to the brain.

Circadian clocks confer adaptation of an organism to environmental variations, including light intensity (20). The major clock resides in the suprachiasmatic nucleus (SCN) of the preoptic area (PA), where the genes of the circadian and circannual rhythms are localised (21, 22). The molecular components involved in the mechanism of the major clock such as CLOCK, BMAL1, and NPAS2 proteins belong to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) family, and are transcription activators of Per genes and nuclear receptors of Rev-erb genes. Protein products of these genes can interact with CO by the heme prosthetic group (23-27). The binding of CO to the transcription factor heme-containing neuronal PAS domain protein (NPAS2) results in the inhibition of DNA-binding activity (23). The concentration of CO affects the affinity of NPAS2 to CO and the ability to form a dimer of NPAS2:BMAL1 (23). An increase in the concentration of CO above 1 µM causes the degradation of the NPAS2:BMAL1 complex (24). Since CO production is synchronised with the circadian rhythm of heme metabolism (23), the interaction of CO with NPAS2 functions as one of the underlying molecular mechanisms of circadian rhythm control (2).

The hypothalamic paraventricular nucleus (PVH) cooperates with the SCN by transmitting light signal (28, 29). The neurons of the PVH exhibit the expression of clock genes (i.e., Per1, Per2) (30) and circadian information is conveyed through the multi-synaptic pathway to the pineal gland (31-34). The function of the PVH is mainly connected with the physiological response to energetic challenges (35).

The current study aimed to test the hypothesis that CO may permeate in the PVC from venous blood to arterial blood and then be transferred by the humoral pathway to the brain, and changes in the concentration of CO in the OphVB may modulate the expression of clock genes and their transcriptional factors in the hypothalamus.

MATERIAL AND METHODS

All procedures were carried out in compliance with Polish legal regulations (act of 21 January 2005), which determined the terms and conditions for experiments on animals, and were in accordance with the protocol of the Local Ethics Commission for Animal Experiments in Lublin No. 8/2007.

Study design

Mature males of a wild boar and pig crossbreed (12 months of age, body mass ~100–120 kg, n=24) were used for the study. The animals were housed in an experimental farm of the Physiology and Reproduction of Animals Department, Rzeszow University in Kolbuszowa, near Rzeszow. They were kept under natural illumination and had ad libitum access to water and standard food. Two groups: control and experimental, with 12 animals each, were used in the study. The experiments were performed during two seasons: during the days with the longest periods of light in the summer (the second half of June) and during the days with the shortest periods of light in the winter (the second half of December). During the summer, the animals were kept in an open-sided shed and exposed to approximately 30,000 lx of natural illumination during the day. The mean ambient temperature was 24°C during the light phase and 12°C during the nocturnal phase. During the winter, the animals were housed in a room with windows and were exposed to between 40 and 50 lx of natural illumination at the eye level of the animals during the day. The mean temperature during the day and night was 12°C. A dim, red spotlight was used to assist in the experimental treatment during the nocturnal phase.

Surgical procedures

The animals were premedicated with atropine (0.05 mg/kg I.M.; Biowet, Gorzow Wielkopolski, Poland) and azaperone (Stresnil 2 mg/kg I.M.; Janssen Pharmaceutica, Beerse, Belgium). General anaesthesia was induced with thiopental sodium (Thiopental, I.V, Sandoz GmbH, Austria). A silastic catheter (o.d., 2.4 mm; i.d., 1.8 mm) was inserted into the right dorsal nasal vein (DNV) toward the OphS. This catheter placement allowed for the infusion of autologous plasma with experimentally elevated concentrations of CO (Fig. 1). An additional catheter (o.d., 2.4 mm; i.d., 1.8 mm) was inserted into the jugular vein (JV) to collect systemic venous blood.

Fig. 1. Schematic drawing of the experiment with infusion of blank autologous plasma (control group) or autologous plasma with ~three-fold elevated concentration of CO (experimental group) into the OphS.

Preparation of autologous plasma with elevated concentrations of carbon monoxide and its infusion in the experimental group

After the surgical procedures, conscious animals were housed in pens where they had free access to food and water and the possibility to change body position. Systemic venous blood was repeatedly collected under sterile conditions from each animal. Heparinised blood (10 IU/ml, Polfa, Poland) was centrifuged (1000×g, 20 min) and plasma was transferred to a sealed glass container (50 ml). Plasma concentration of CO in the OphVB was estimated using a standard addition method (8). The average concentration was 4.03 nmol/ml and 0.89 nmol/ml in June and December, respectively. Plasma was supplemented with chromatographically pure CO (0.8 cm3 to each portion) and stirred with a roller for 30 min, and the concentration of CO was measured again. The autologous plasma, with increased concentrations of CO, up to 13.54 nmol/ml in June and 3.13 nmol/ml in December, was infused at a rate of 8.3 ml/h with the use of a pump (SEP 21S, Ascor, Poland) for 48 hours into the OphS (Fig. 1), from which the venous blood flowed into the VCS of the PVC. The autologous blood cells remaining after the collection of plasma were mixed with Ringer solution in a volume equivalent to collected plasma. The suspension was stirred with a roller for 30 min and then continuously infused into the JV. Autologous blood plasma (blank) was infused into the OphVS of the control group at a rate of 8.3 ml/h.

Preparation of the preoptic area and dorsal hypothalamus tissue for total RNA isolation

After the end of infusion of the autologous blank plasma or plasma with elevated CO concentrations, the animals of the control and experimental groups were transported to a properly adapted slaughterhouse. Both groups were transported and sacrificed during the summer day (n=3), during the summer night (n=3), during the winter day (n=3) or during the winter night (n=3). The animals were sacrificed by electrical stunning and exsanguination. Then, the head was immediately cut and the skull was opened, the hypothalamus was isolated from the brain; the PA and DH tissues were collected and shock-frozen in liquid nitrogen (–196°C). Samples were stored at –70°C until required for analysis.

Total RNA isolation

Ice-cold brain tissues suspended in 1 ml of Tri-Reagent solution (Molecular Research Center, Cincinnati, OH, USA) in RNase free tubes with ceramic beads were homogenized in a FastPrep-24 apparatus (MP Biomedicals LLC, Solon, OH, USA) for 45 s. Following homogenisation, total RNA was isolated using previously described method (36). Total RNA was isolated from the chloroform fraction using a kit for total RNA (A&A Biotechnology, Gdansk, Poland).

RNA integrity was checked by 2% agarose gel electrophoresis. The quality was expressed as the absorbance ratios at 260:280 nm and the amount of RNA in the sample was measured using a Nano Drop (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Real-time PCR analysis

Real-time PCR was performed using a Maxima First Strand cDNA kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and all steps were conducted according to the manufacturer’s instructions. Briefly, aliquots of RNA sample (1 µg) were treated with RNase-free DNase for 10 min at room temperature. The treated RNA samples were denatured for 10 min at 70°C prior to synthesis of complementary DNA. For first-strand cDNA synthesis, both oligo dT and random hexamers (50 pmol) were used. The reaction mixture contained 10 mM dNTP mix, 200 U Reverse Transcriptase M-MuLV RNase H Minus in a 5×RT buffer (250 mM Tris-HCl, 250 mM KCl, 20 mM MgCl, 20 mM DTT, pH 8.3 at 25°C), and 20 U RiboLock RNase inhibitor. cDNA synthesis was carried out in a PCR Thermal Cycler (Bio-Rad, Hercules, CA, USA) according to the following thermal cycle program: 25°C for 15 min, 50°C for 25 min, 85°C for 5 min, and then held at 4°C. Each sample was used in duplicate. Following analysis, aliquots of the RT products (ssDNA) were diluted 20-fold with nuclease-free water and used for real-time PCR analysis, as described below.

The ssDNA was subjected to quantitative PCR analysis, using a real-time PCR system (ABI 7900HT; Applied Biosystems, Foster City, CA, USA). Each ssDNA sample was assayed in duplicate. The reaction mixture contained: 5 µl template (equivalent to 12.5 ng RNA), 5 µl primers (forward and reverse), 2.5 µl TaqMan probe, and 12.5 µl Maxima probe PCR mix (Thermo Fisher Scientific Inc., Waltham, MA, USA). Thermal cycling was initiated at 95°C for 10 min for DNA polymerase activation. Forty steps of PCR were performed, each one consisting of heating at 95°C for 15 s and 60°C for 60 s. All primers and probes was designed using Primer Express Software v3.0 (Applied Biosystems, Foster City, CA, USA) (Table 1). Relative gene expression was calculated by comparing the genes of interest with reference gene (Sdha) and was expressed in arbitrary units. The selection of the reference gene was carried out based on published data (37). For result calculation, the real time PCR Miner algorithm was used (38).

Table 1. The primers and probes used.

Statistical analysis

Statistical analysis was performed by one-way ANOVA with Bonferroni post test for selected pairs of columns. A critical value for significance in all experiments was P<0.05. For statistical analysis, GraphPad PRISM software version 5.00 for Windows (San Diego, CA, USA) was used. All data are shown as a box and whiskers min. to max. Values are means ±S.E.M.

RESULTS

The expression of clock genes and their regulators in the preoptic area and dorsal hypothalamus

The expression of clock genes Per1, Per2, Cry1, Cry2 and Rev-erb α, Rev-erb β, and the genes of their regulators Bmal1, Clock, Npas2 and Ror β was detected in two areas involved in the reception and transmission of light signal, i.e., in the PA and DH in males of a wild boar and domestic pig crossbreed. Table 2 presents the comparison of expression level of these factors in these two areas of the hypothalamus in the control group. The expression of clock genes Per1, Cry1, Cry2 and Rev-erb β was higher, while Per2 was lower in the DH than in the PA during the day and night in both seasons. The particularly high level of Cry1 expression in the DH is noteworthy. The expression of the regulatory genes Bmal1, Clock, and Npas2 did not differ between the structures in any of the tested periods in control animals. Rev-erb α and Ror β remained constant in the DH in most examined periods, except in the winter night (Rev-erb α) and in the summer day (Ror β), when their expression was significantly higher.

Table 2. Comparison of basal gene expression in the PA and DH at 12-hour intervals (always in the middle of subjective day and night in two seasons: winter and summer).

The influence of carbon monoxide on the expression of clock genes and regulatory genes in the preoptic area and dorsal hypothalamus

The expression of the clock genes Per1, Per2, Cry1, Cry2 and Rev-erb α, Rev-erb β, and regulatory genes Bmal1, Clock, Npas2 and Ror β in the PA and DH in animals infused with autologous plasma with experimentally elevated concentrations of CO (experimental group) was compared with the control group. The level of expression of the clock genes Per1 and Cry2 in the PA was higher in the experimental group than in the control group, both in the summer and winter and during the day (P<0.01) and night (P<0.001), and the expression of Per2 in the PA was lower during the winter day (P<0.001) (Fig. 2). In the PA, Rev-erb α expression was lower in the winter night (P<0.05) and in the summer day (P<0.001) in animals treated with CO than in the control group. Rev-erb β expression was higher after CO treatment in the winter day and night (P<0.01 and P<0.05), but in the summer the increase of this gene expression occured only at night (P<0.05) (Fig. 6 panel A and B). In the DH, the expression of the genes Per1 and Per 2 in experimental group was higher than in control in both seasons during the day (P<0.01) and night (P<0.001). The expression of Cry1 in the DH was lower during the winter day (P<0.05) and summer night (P<0.001), and Cry2 expression was lower during the winter day (P<0.01), and during the summer day (P<0.001) and night (P<0.01) in experimental group versus control group (Fig. 3). Rev-erb α and Rev-erb β expression in the DH was lower in the experimental group in the winter night (P<0.05) and Rev-erb β expression was lower in the summer night (P<0.05) in comparison to the control group (Fig. 7 panel A and B).

Fig. 2. The expression of four clock genes: (A), Per1; (B), Per2; (C), Cry1; (D), Cry2 in the PA of the brain. Transparent boxes show the control group (n=3); grey boxes show the CO-treated group (n=3). Statistical significance is marked as follows: * P<0.05, ** P<0.01, *** P<0.001.

Fig. 3. The expression of four clock genes: (A), Per1; (B), Per2; (C), Cry1; (D), Cry2 in the DH of the brain. Transparent boxes show the control group (n=3); grey boxes show the CO-treated group (n=3). Statistical significance is marked as follows: * P<0.05, ** P<0.01, *** P<0.001.

In the PA, the expression of Bmal1 in the experimental group was lower in both seasons during the day and night (P<0.001), the expression of Clock was lower during the summer day and night (P<0.05), and the expression of Npas2 was lower in both seasons during the day (P<0.01) and night (P<0.001), compared with the control group (Fig. 4). The Ror β expression level was lower after CO treatment in the winter day and night (P<0.01, P<0.05) and in the summer night (P<0.001) than in the control (Fig. 6 panel C). In the DH, the expression of all three regulatory genes Bmal1, Clock, and Npas2 in the experimental group in both seasons was lower during the day (P<0.001) and night (P<0.001) than in the control group (Fig. 5). The level of Ror β expression was lower in the experimental group in winter day (P<0.01) and in the summer day and night (P<0.001) compared to the control (Fig. 7 panel C).

Fig. 4. The expression of clock gene transactivators: (A), Bmal1; (B), Clock; (C), Npas2 in the PA of the brain. Transparent boxes show the control group (n=3); grey boxes show the CO-treated group (n=3). Statistical significance is marked as follows: * P<0.05, ** P<0.01, *** P<0.001.

Fig. 5. The expression of clock genes transactivators: (A), Bmal1; (B), Clock; (C), Npas2 in the DH of the brain. Transparent boxes show the control group (n=3); grey boxes show the CO-treated group (n=3). Statistical significance is marked as follows: * P<0.05, ** P<0.01, *** P<0.001.

Fig. 6. The expression of clock genes (nuclear receptors): (A), Rev-erb α; (B), Rev-erb β; (C), Ror β in the PA of the brain. Transparent boxes show the control group (n=3); grey boxes show the CO-treated group (n=3). Statistical significance is marked as follows: * P<0.05, ** P<0.01, *** P<0.001.

Fig. 7. The expression of clock genes (nuclear receptors) : (A), Rev-erb α; (B), Rev-erb β; (C), Ror β in the DH of the brain. Transparent boxes show the control group (n=3); grey boxes show the CO-treated group (n=3). Statistical significance is marked as follows: * P<0.05, ** P<0.01, *** P<0.001.

DISCUSSION

In the current study, the concentration of CO in venous ophthalmic blood in experimental animals was approximately three times higher compared with the control animals. We demonstrated that the expression of clock genes and the genes of their regulatory factors was altered in the experimental group compared with the control group both in the PA and DH. The current study also demonstrated that the level of normal expression of genes that form a functional intrinsic biological clock differed between the studied structures of the hypothalamus. The response to an increased concentration of CO differed between individual genes and the hypothalamic regions. However, Per1 was the only gene whose expression was increased in animals treated with CO compared with control both in the PA and DH, and regardless of the time of day and season. It has been established that increased concentrations of CO may modulate the interaction of particular components involved in the molecular mechanism of the biological clock controlling most physiological and behavioural functions in mammals (2). This enables us to consider that the increased supply of CO to the hypothalamus could be the factor stimulating the expression of the Per1 gene in our experimental animals. In addition, a study performed in an identical animal model demonstrated that changes in CO concentration in the OphVB could have an impact on the protein levels of the melatonin synthesis pathway enzyme arylalkylamine N-acetyltransferase, with parallel changes in systemic melatonin levels (Romerowicz-Misiak et al., unpublished observation). Thus, our hypothesis on the possibility of the countercurrent transfer of CO in the PVC and the supply of CO to the brain by the humoral pathway, being the basic problem of current research, seems reasonable.

Many authors believe that Per1 is clearly regulated by light (39, 40). The current results demonstrate that the level of Per1 expression in the PA and DH was elevated in the animals treated with CO. Our earlier study demonstrated that the amount of CO released from the eye to venous blood under the condition of intensive natural illumination during the summer day was several times higher than that during the night and winter day and night (8). The above data together may confirm the concept of humoral phototransduction, published earlier by Oren (19), assuming that the CO produced in the eye when exposed to light can be a transmitter of light signal.

The function of two hypothalamic structures, the PA and DH, is related to the reception and transmission of light signal. Per1 is considered a crucial element of the clock mechanism, responsible for a rapid response to light signal (41). In the current study, a prolonged infusion of plasma with elevated CO concentration imitated long exposure to the light. In both studied structures (the PA and DH), Per1 responded to CO by its increased expression and we believe that this points to the role of Per1 as a specialised factor for the input of light signals. As mentioned above, in our study, the increase in Per1 expression level was especially notable in the PA and it could be assumed that this resulted in the resetting of the whole regulatory loop of the clock. Similarly, the stimulation of Per1 expression was observed in the liver cells of mice treated with heme (42). This effect was particularly noticeable at night. The authors supposed that the increase in gene expression could be due to the products of heme degradation, among others CO, regarded by us as a humoral signal for light. The profile of clock gene expression, particularly Per1, presented by these authors is similar to that observed in the current study. In addition, the authors believed that Per1 protein increased the activity of the promoter of its gene, activated by BMAL1/NPAS2 (42). The existence of a constitutive connection of the BMAL1/CLOCK dimer to Per1 E-boxes has also been shown (43). Since Per1 knockout mice could not be entrained to a long photoperiod, it has been postulated that Per1 is important for shifting the phase to a long photoperiod (44). This process is related to the Per2 gene and expression of its protein product. Long exposure to light of Per1 knockout mice changed the levels of Per2 protein but did not alter the levels of mRNA of the gene (44). The expression of Per2 did not change in the PA after CO treatment in our study, but it seems that it had lost its oscillatory profile. In contrast, the expression of Per2 was increased in the DH after CO treatment. Therefore, it may be assumed that Per2 plays a different role depending on its location. The presence of Per2 in the DH, the structure with a role in light signal transmission, and the increase in its expression in this structure after CO treatment (regarded by us as a humoral carrier of light signal) might suggest the role of this gene as an output light signal factor, which can link the intrinsic clock with physiological pathways (45). Regarding darkness signaling, the treatment with melatonin resulted in a reduction of Per2 expression in the rat PVH (30). There are divergent opinions on the participation of Per1 and Per2 genes in the functioning of the biological clock mechanism. We suggest that these two genes are equally important for the development of proper functioning of negative and positive feedback loops of the circadian clock in the SCN. The reduction in Bmal1, Clock, and Npas2 expression observed in the current study may be explained on the basis of the overexpression of Per1 (44). The function of Cry genes under the conditions of our experiment is unclear. It is possible that the response of these genes would be more clear if the profiles of their protein products were evaluated. However, the determination of the concentration of these proteins in the nuclear fraction of the examined structures of the hypothalamus was not possible in our study due to the small size of the investigated structures. We think that the molecular profile shown in our results indicates that the positive limb of the circadian feedback loop was dampened and the negative part of this loop was enhanced. It is known that CRY proteins are essential components of the negative feedback loop (46) and are necessary for the maintenance of circadian rhythms (47). It has been shown that in Cry2–/– mice, the sensitivity of the Per1 gene to acute light induction in the SCN is reduced and it has been reported that cryptochromes may function as photoreceptive proteins (48, 49). The results of current study in which CO was considered by us as the mediator of light signal that caused the high expression of Per1 accompanied by high expression of Cry2 seem to confirm the findings of the abovementioned authors on the role of Cry2 in phototransduction. In addition, the lack of effect of CO in the DH might confirm the role of Cry genes as essential elements of the clock mechanism located in the SCN, related to light/dark induction.

Rev-erb’s and Ror β are nuclear receptors involved in Bmal1 regulation. They create the second circadian loop interlocked with the Per/Cry loop. Both of them act and compete on RORE motifs in Bmal1 gene promoter. Rev-erb’s repress Bmal1 gene expression while Ror β activate it (50, 51). Our results demonstrate that, carbon monoxide alters the expression of Rev-erb’s and Ror β mostly in PA and modulates the regulation of the circadian clock. As demonstrated in our study, these findings are consistent with the low level of Bmal1 expression after CO treatment. Our study revealed that low Ror β expression indicates a lack of activated Bmal1 expression via RORE elements in Bmal1 promoter and low expression of this gene. Additionally, we showed that high Rev-erb β expression in PA sustains or enhances low Bmal1 expression through the repression of this gene promoter. Furthermore, low Ror β and Bmal1 expression was also observed in DH, but not in that of Rev-erb’s, in comparison to the control. Interestingly, the decreased Bmal1 expression is not so distinct in DH compared to PA, which indicates that Rev-erb’s may sustain or enhance the negative effect of decreased Ror β expression.

Studies on the expression of clock genes have so far been carried out almost exclusively with the use of commercially available cell lines or in small experimental animals. The present study was performed in a crossbreed of the domestic pig and wild boar, species in which, to our knowledge, such studies have not been conducted. The current results raise many questions and, therefore, must be considered to be preliminary research. The study will be continued.

In conclusion, this study demonstrated the influence of elevated concentrations of CO in OphVB on the expression level of clock genes in the PA and DH in a domestic pig and wild boar crossbreed. These results confirmed the hypothesis on the ability of CO to function in humoral transfer from the eye to the structures involved in the reception and transmission of light signal and the effect of CO on clock gene expression. Furthermore, our results indicate that our experimental animals after CO treatment have their master clock machinery deregulated which could cause chronodisruption. This phenomenon may have many negative implications in peripheral organs such as the liver or gastrointestinal tract which are tuned to central pacemaker in SCN (53, 54).

Abbreviations: CO, carbon monoxide; DH, dorsal hypothalamus; DNV, dorsal nasal vein; HO, heme oxygenase; ICA, internal carotid artery; JV, jugular vein; OphS, ophthalmic sinus; OphVB, ophthalmic venous blood; PA, preoptic area; PVC, perihypophyseal vascular complex; PER-ARNT-SIM (bHLH-PAS), basic helix-loop-helix; PVH, paraventricular nucleus; SCN, suprachiasmatic nucleus; VCS; venous cavernous sinus.

Acknowledgements: This study was supported by the Polish State Committee for Science Research (N N 311 1001 33) and the Ministry of Science and Higher Education in 2011 and 2012.

The data were previously presented during IX Symposium: Genetic, Environmental and Physiological Conditionalities of Reproduction, Animal Health and Safety and Quality of Animal Origin Food; Wierzba, 16–17 May 2012. P. Gilun, M. Koziorowski, S. Stefanczyk-Krzymowska. Influence of seasonal and diurnal changes of light transmitted by humoral pathway on the regulation of biological clock gene expression and secretion of hormones reproductive processes.

Author contributions: Conception and design of the experiments: P.G., S.S-K. and M.K. Collection, analysis and interpretation of data: all the authors. Drafting the article: P.G., S. S-K. Revising it critically for important intellectual content: all the authors.

Conflict of interests: None declared.

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