Circadian heme oxygenase actitivy in the hamster suprachiasmatic nuclei

Neuroscience Letters 353 (2003) 9–12 www.elsevier.com/locate/neulet

Circadian heme oxygenase actitivy in the hamster suprachiasmatic nuclei Marı´a Fernanda Rubio, Patricia V. Agostino, Gabriela A. Ferreyra, Diego A. Golombek* Departamento de Ciencia y Tecnologı´a, Universidad Nacional de Quilmes, R.S. Pen˜a 180, Bernal, 1876 Buenos Aires, Argentina Received 16 April 2003; received in revised form 14 August 2003; accepted 18 August 2003

Abstract Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the hypothalamic suprachiasmatic nuclei (SCN). We have tested the participation of heme oxygenase (HO) in the SCN, by assessing HO specific activity at different time points and photic conditions. HO activity was determined by the conversion of hemin to bilirubin. HO enzymatic activity in the SCN was significantly higher during the night than during the day; this difference persisted when animals were placed under constant darkness, suggesting an endogenous circadian control. HO inhibition by Zn-protoporphyrin did not affect light-induced phase shifts in vivo, suggesting that the enzyme is not necessary for light input to the clock. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Circadian; Suprachiasmatic; Entrainment; Heme oxygenase; Carbon monoxide; Hamster

Mammalian circadian rhythms are under the dual control of the environment and an endogenous clock located in the hypothalamic suprachiasmatic nuclei (SCN). Free-running rhythms are entrained by means of light-induced phase shifts that reach the SCN through a glutamatergic retinohypothalamic tract [11]. Glutamate interacts with Nmethyl-D -aspartate (NMDA) and non-NMDA receptors in the SCN, and induces a cascade of events that results in a chronic phase shift of clock-controlled rhythms [11]. Ca2þ and cyclic GMP (cGMP)-related signal transduction pathways have been reported to play a fundamental role in circadian entrainment [11], ultimately leading to changes in the expression and/or activity of specific clock genes [16]. A role for nitric oxide (NO) in circadian responses to light has been suggested [3]. One of the targets for this messenger is the activation of a soluble guanylyl cyclase (GC) in the SCN, resulting in an increase of cGMP levels. However, other signals might also be responsible for cGMP formation in the SCN. In 1991 Marks et al. [13] postulated that carbon monoxide (CO, whose synthesis depends upon the activation of heme oxygenase) shares some of the chemical and biological properties of NO, by binding to the heme moiety of soluble GC to produce cGMP [12]. Heme oxygenase (HO) is a microsomal enzyme that oxidatively * Corresponding author. Tel.: þ54-11-4365-7100x154; fax: þ 54-114365-7132. E-mail address: [email protected] (D.A. Golombek).

cleaves the heme ring to form biliverdin, ferrous iron, and carbon monoxide (CO). Two principal forms of HO (HO-1 and HO-2) have been isolated, characterized and cloned [2]. HO-3 is a more recently identified isoform with low activity [14]. Despite their close functional similarity, these proteins are the products of different genes and they exhibit differences in their sizes, biochemical characteristics, antigenicity, and tissue distribution. HO-1 can be induced in spleen and liver by a variety of stressful conditions, including exposure to heavy metals, oxidative stress, inflammatory cytokines, and heat stress [12]. HO-2 is widely distributed throughout the body, and is responsible for most of HO activity in the brain [7,12], where it plays a neuromodulatory activity, and its product, CO, has been shown to be a putative neurotransmitter [2,12,18]. HO-2 has been recently found in rat SCN neurons and has been reported to play a role in clock resetting by cholinergic agents [1]. Moreover, it has been recently reported that the transcription factor NPAS2 (neuronal PAS domain 2), a member of the molecular machinery that regulates circadian rhythmicity, is itself a hemoprotein whose transcriptional activity is regulated by CO [4]. The aim of this study was to analyze HO presence and activity in the hamster SCN and its putative role in circadian photic entrainment. Syrian hamsters (Mesocricetus auratus) were raised in our colony and housed under a 14:10-h light/dark cycle (L:D), with food and water ad libitum. When animals had to be

0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.08.075

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killed in the dark, we used a dim red light source (5 lux). Under constant dark conditions (D:D), circadian time 12 (CT12) was defined as the onset of wheel-running activity. For the determination of enzymatic activity, animals were killed at 12:00 h (ZT4) or 24:00 h (ZT16). To determine the effect of light on HO activity, animals were moved into light or darkness for 2 h before ZT16 or ZT4, respectively. For circadian determinations, animals were moved into constant darkness for 48 h and killed at CT4 and CT16. Animals were killed by decapitation, and their brains were excised and placed in an ice-cold environment. A 500mm hypothalamic slice was cut and tissue containing the SCN was punched out. HO activity was determined by the method of Ewing et al. [6] slightly modified. Unless stated otherwise, all drugs came from Sigma (St. Louis, MO). Tissues were mechanically disrupted with a glass rod in 50 mM ice-cold phosphate buffer (pH 7.4) containing 0.25 M sucrose and protease inhibitors, and centrifuged at 203 £ g for 10 min. The supernatant was centrifuged at 9000 £ g for 20 min, and the resulting supernatant was centrifuged at 105 000 £ g for 1 h. The pellet was resuspended in 50 mM phosphate buffer (pH 7.4) containing protease inhibitors. The microsomal supernatant fraction from the liver of a rat served as the source of biliverdin reductase. Bilirubin formation was catalyzed in a reaction mixture (200 ml) containing hemin (200 mM), 10 ml rat liver microsomal supernatant fraction (0.5 mg/ml), MgCl2 (0.2 mM) and a NADPH-generation system containing NADPH (1.33 mg/ml), glucose-6-phosphate (2 mM) and glucose-6-phosphate dehydrogenase (1 U/ml) in phosphate buffer (pH 7.4, 50 mM). The reaction was conducted for 2 h at 37 8C in the dark and stopped by placement at 2 20 8C, after which 0.6 ml of chloroform were added; after the extraction, the chloroform layer was spectrophotometrically measured. Bilirubin formation was calculated from the difference in absorption between 464 and 530 nm using a millimolar absorption coefficient of 40 mM21 cm21. Blank samples contained either the reactants without the tissue or boiled tissue. To determine the presence of HO-1 and HO-2 in the hamster SCN, animals were killed at ZT4 and ZT16, their brains were quickly excised on ice and SCN tissue punched out and homogenized in 50 mM Tris – HCl buffer (pH 7.4) containing protease inhibitors. SCN proteins (20 mg) were electrophoretically transferred overnight (30 V) to nitrocellulose membranes. After preincubation with blocking buffer (Tween-20 0.1% v/v in Tris-buffered saline (pH 7.4) (TTBS), with blocking milk 10%), the nitrocellulose membranes were incubated 1.5 h at room temperature with HO-1 or HO-2 antibodies (Santa Cruz, CA, diluted 1:10 000). Membranes were washed with TTBS and immunoreactivity for HO-1 and HO-2 was assessed using a secondary coupled to horseradish peroxidase (Santa Cruz), and visualized with the ECL system (Amersham). To determine the participation of HO in light-induced phase advances, hamsters were anesthetized with 75 mg/kg ketamine and 10 mg/kg xylazine and implanted with 22-

gauge stainless steel guide cannulae (Plastics One, Roanoke, VA) aimed for the third ventricle. Cannulae were implanted 1.0 mm above the target site (coordinates relative to bregma: anteroposterior þ 0.6 mm, dorsoventral 2 8.2 mm, mediolateral 0.0 mm, tooth bar was set at 2 2.0 mm). After recovery from the anesthesia, hamsters were individually housed in cages equipped with running wheels and then moved to D:D. Wheel-running activity was monitored continuously and recorded using the Dataquest III system (Minimitter Co. Inc.). The onset of activity for each day or circadian cycle was used as a marker of the activity rhythm and was defined as CT12. Phase shifts in D:D conditions were calculated using eye-fitted lines drawn through consecutive onsets of activity for the 7 days before each experimental manipulation (day 0) and on subsequent days 4 through 10. These were used to extrapolate CT12 on day 1 (day following manipulation). The phase shift, estimated by three independent observers, was defined as the difference between the timing for CT12 on day 1 projected by pre- and postpulse lines. Fifteen minutes before a 10-min light pulse (150 lux) at CT18, animals received a microinjection of 10 mg or 50 mg Zn-protoporphyrin-IX (ZnPP IX, diluted in 1 ml dimethylsulfoxide (DMSO)/saline 50%/50%), an HO inhibitor, or vehicle, over a 5-min period. Control animals received drug or vehicle injections without light pulses at CT18. The dose of ZnPP was chosen according to the literature (i.e. [10]), while the timing (i.e. 15 min) of the manipulation before the light pulse was chosen so that it would parallel the effect of NOS inhibitors on light-induced phase changes [3,15], under the hypothesis that the time course of activation/ inhibition for both enzymes is similar. As shown in Fig. 1A, hamster SCN HO activity exhibited a significant diurnal variation when assessed during the light and dark phases of the daily photoperiod, peaking at night. These variations persisted under constant dark conditions, suggesting an endogenous origin. SCN HO activity probably reflects the presence of the HO-2 isoform alone, since we found HO-2 but not HO-1 in this tissue (Fig. 1B). The diurnal variation in HO activity does not seem to be caused by temporal changes in the amount of enzyme, since Western blot of soluble SCN proteins harvested at both time points (ZT 4 and ZT 16), indicated no differences between diurnal and nocturnal HO-2 levels (Fig. 1B). HO-1 was not detected in the hamster SCN at any time. We also tested the ability of light to modify HO activity by evaluating the effect of 2 h of light (at night) or darkness (during the day) under L:D conditions. As shown in Fig. 1C, no significant changes were found after these stimuli. To determine whether intracerebroventricular (i.c.v.) administration of an HO inhibitor could attenuate lightinduced phase advances of the locomotor activity rhythms, animals were injected with ZnPP IX (10 or 50 mg, i.c.v.) or vehicle 15 min before a 10-min light pulse (150 lux) at CT18. As shown in Fig. 2, this drug had no effect on light-induced circadian phase changes at CT18 (vehicle þ light: 60.8 ^ 21.9 min; 10 mg ZnPP IX þ light: 65.6 ^ 19.9 min).

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Fig. 2. Effects of ZnPP IX on light-induced phase shifts at CT18. (A) Representative actograms of hamster wheel-running activity. The arrows indicate the day of treatment. Left panel: representative actogram of an animal receiving an i.c.v. ZnPP IX (10 mg) administration followed by a light pulse at CT18; right panel: representative actogram of an animal receiving vehicle i.c.v. before a light pulse at CT18. (B) Summary of lightinduced phase changes at CT18 (see text for details). Fig. 1. (A) Diurnal and circadian variation of HO enzymatic activity in the hamster SCN. Animals were sacrificed at ZT4 or ZT16. HO activity was significantly higher during the night (*P , 0:05, Student’s t-test). In another set of experiments, animals were killed after 48 h under D:D, at CT4 or CT16, showing maximal values of HO activity during the subjective night (**P , 0:001, Student’s t-test). (B) Western blot of HO levels in the SCN. Hamsters were killed at ZT4 or ZT16. SCN proteins (20 mg) were electrophoretically transferred and incubated with a specific HO-2 antibody, evidencing an immunoreactive band that did not exhibit differences between both time points tested. Liver and whole brain were used as negative and positive tissue controls, respectively. (C) Effect of 2 h dark or light pulses delivered to hamsters under L:D at ZT4 or ZT16, respectively, on SCN HO activity levels. HO activity was not affected by photic stimuli.

As mentioned above, the presence of HO has been demonstrated in the rat SCN by immunohistochemistry [1]. In the present work HO activity was assessed in the hamster SCN, by employing a spectrophotometric assay that has been successfully used for the measurement of this activity in several systems [5]. The average value of HO activity assessed in the SCN was in the range of nmol/mg protein/h, close to the levels detected in other structures like rat spleen, liver, and kidney [5,10]. In addition, present results demonstrate that HO-2, but not HO-1, is expressed in this tissue, as shown by western

blotting analysis. However, this negative result does not formally rule out the possibility that this isoform may also be expressed in this tissue in response to appropriate stimuli. Although no changes in HO-2 levels were found among the time points tested, SCN HO activity was significantly higher at midnight than midday. In constant darkness, day – night variation persisted. These results, together with the demonstration that light stimuli did not affect SCN HO activity, support the existence of a circadian clockcontrolled function. The intracellular events that control the changes in HO activity remain to be established. A single glucocorticoid response element (GRE) is present and functional in the HO-2 gene [12]; in addition, phosphorylation of HO-2 through a protein kinase C (PKC)-related pathway has been shown to enhance its catalytic activity [2]. HO seems to be under the output control of the circadian clock. We also evaluated the possibility thpat HO activity is involved in the input mechanism for circadian entrainment, by pharmacological blockade of the enzyme prior to a light pulse at CT 18, a time when this stimulus induces phase advances of rhythms. Administration of ZnPP had no effect

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on photic phase shifting, suggesting that HO activity is not required for light input into the clock, although this possibility cannot be ruled out at present. This result seems consistent with the demonstration the light stimuli did not affect SCN HO activity. Other entraining stimuli that act upon the circadian clock include cholinergic agents, which interact with SCN muscarinic receptors and cGMP-related signaling to phase advance overt rhythms. In vitro, pharmacological inhibition of HO block muscarinic-induced resetting, while hemin (a HO activator) induced phase shifts, suggesting that acetylcholine photic-like pathways are mediated by carbon monoxide in the SCN [1]. Moreover, nitric oxide synthase (NOS) inhibition indicated that NO is involved in the same pathway as CO [1]. In a previous report, we demonstrated that SCN NOS activity had a day-night variation with a nocturnal increase [8], that could be related to changes in the endogenous modulators of this enzyme. In addition, NOS inhibition results in a partial blockade of light effects on circadian entrainment [3,8,15]. The gaseous product of NOS, nitric oxide (NO), is thus not only regulated in a diurnal fashion, but could also be involved in zeitgeber input. Despite the notable similarities between NOS and HO [2], we have found two fundamental differences between the regulation of HO and NOS in the SCN: (1) NOS activity is regulated on a diurnal, but not circadian, basis, while HO activity varies under both L:D and constant conditions, and (2) NOS, but not HO, activity, is at least in part required for light-induced phase changes. As for downstream mechanisms that could be the targets of these enzymes, it has been shown that both NO and CO affect guanylyl cyclase, resulting in an increase of cGMP levels, a nucleotide that has been involved in circadian phase advances to light [9,17]. The relative contribution of each of these gaseous messengers remains to be established. Very recently, Dioum et al. [4] showed that CO is able to regulate transcriptional activity in the circadian clock by binding to a gas-responsive sensor in the clock gene NPAS2, which, when dimerized with BMAL1 controls circadian rhythmicity in several tissues. Moreover, genes encoding rate-limiting enzymes for heme biosynthesis are under strict circadian control [19], regulated by clock genes period 1 and period 2, indicating that heme metabolism in general is a target of the circadian machinery. In summary, in this paper we open the possibility of a clock-regulated heme oxygenase activity that could serve as part of the output pathways through which the circadian oscillator fine-tunes body rhythms into a coherent internal temporal order.

Acknowledgements Supported by ANPCyT, CONICET, UNQ and Fundacio´n Antorchas (Argentina). We are grateful to Ruth Rosenstein

for critical reading of the manuscript, and Marı´a Ine´s Keller Sarmiento and Cora Cymmering for expert advice and technical assistance.

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