Seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, Mytilus edulis ISJ 7: 141-145, 2010 ISSN 1824-307X RESEARCH REPORT Seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, Mytilus edulis: temperature controls physiological processes KJ Mantione, P Cadet, F Casares, W Zhu, GB Stefano Neuroscience Research Institute, State University of New York at Old Westbury, NY 11568, USA Accepted May 13, 2010 Abstract It is anticipated that invertebrate processes will be subject to seasonal variations because of their poikilothermal characteristics. In the present study we determined if the morphine coupled nitric oxide (NO) release, which is constitutive in nature, exhibits seasonal characteristics, which has previously been shown for catecholamine processes in the marine mollusc Mytilus edulis. In this regard, morphine induced NO release measured on a monthly basis for one year revealed a peak release value (39 ± 4 nM) during the late spring and early summer. The lowest NO release occurred during the months of January (6.0 ± 0.5 nM) through March (6.5 ± 1.1 nM). The lowest sea surface temperatures (1.3 °C) were also recorded in these same three winter months in New York. Relative mu opiate receptor gene expression was assessed by real time PCR during these seasons. The mRNA expression reached a relative peak during the month of June and was at its lowest in February and March, further demonstrating the direct coupling of morphine with this receptor. We conclude that the temperature an animal is chronically exposed to serves to control cellular processes, i.e., opiate signaling. Key Words: nitric oxide; mu opiate receptor; morphine Introduction Through homeostasis, living organisms maintain their survival in the face of both internally and externally generated stimuli. This balance is constantly challenged and therefore the ability to overcome these normal perturbations is essential to survival and longevity (Chrousos and Gold, 1992; Fricchione and Stefano, 1994). In this regard, subjecting invertebrate animals to temperature changes has been shown to alter the ganglionic monoamine levels as well as affecting functionality of gill cilia in Mytilus edulis (Stefano et al., 1977a, 1977b; Stefano and Catapane, 1977b). Additionally, opiate processes respond to various types of ___________________________________________________________________________ Corresponding author: Kirk J Mantione Neuroscience Research Institute SUNY - College at Old Westbury P.O. Box 210 Old Westbury, NY 11568-0210, USA E-mail: KJMantione@sunynri.org List of abbreviations: Nitric oxide, NO; phosphate buffered saline, PBS; S-nitroso-N-acetyl-DL-penicillamine, SNAP; constitutive nitric oxide synthase, cNOS stressors in both vertebrates and invertebrates ( Lee and Spector, 1991; Marrazzi et al., 1997; Sonetti et al., 1999; Zhu et al., 2001; Cadet et al., 2002; Guarna et al., 2002). We have previously demonstrated that endogenous morphine represents the terminal component of a successful stress response and that its actions are generally down regulating immune responses and metabolic rates (Stefano et al., 2000). This down regulation occurs because of coupling of the mu opiate receptor to nitric oxide (NO) release (Cadet et al., 2003). Our group has also demonstrated that opiate receptors and subsequent morphine induced NO release can be profoundly impacted by temperature changes in M. edulis (Cadet et al., 2002; Mantione et al., 2003). Furthermore, we have presented molecular evidence on the effect of rapid temperature changes on mu opiate receptor expression and morphine levels in this invertebrate’s nervous system (Cadet et al., 2002). In cold stressed organisms, ganglionic mu opiate receptor decreases and morphine levels increase (Cadet et al., 2002). In the present study, we investigated the seasonal variation in ganglionic mu opiate receptor expression in M. edulis. In addition, we performed a morphine induced NO release assay to determine the functionality of the morphine signaling system in this model organism. 141 Month Jan FebMarchApril May June July Aug Sept Oct Nov Dec nM N itr ic O xi de 0 10 20 30 40 50 T em pe ra tu re o C 0 10 20 30 40 50 [NO] Sea surface temperature Fig. 1 Monthly measurements of sea surface temperature and morphine (1 µM) stimulated NO release from Mytilus edulis pedal ganglia (20 ganglia per assay, n = 4). Paired t-tests revealed statistically significant differences between January or February or March and May or June or July (p = 0.002). Material and Methods Animal collection and Nitric Oxide (NO) determination Mytilus edulis collected from Shinnecock Bay on Long Island were immediately transported to the laboratory in seawater for processing. The ambient seawater temperature was maintained using insulated coolers until dissection of animals. Samples were collected monthly on the 15 day of the month and sea surface temperature was recorded. For NO determination, approximately 20 pedal ganglia were placed in 1 ml phosphate buffered saline (PBS) at room temperature. NO release from the tissues was immediately directly measured using a 200 μm flexible NO-specific amperometric probe (World Precision Instruments, Sarasota, FL) connected to a 4-channel Biostat (ESA, Chelmsford, MA). The system was calibrated daily with S-nitroso-N-acetyl-DL-penicillamine (SNAP) in 0.1 M Cu+2. The amperometric probe was allowed to equilibrate in PBS for at least 10 min prior to being transferred to the tube containing the tissue. Morphine-stimulated NO release was evaluated at a final concentration of 10-6 M. Each experiment was repeated four times (4 groups of 20 ganglia for each month) along with a control (PBS only). A paired student’s t-test was performed to evaluate differences between selected months. Mu opiate receptor expression Pedal ganglia (15) were immediately processed after dissection. The ganglia were placed in 1.5 ml tubes and then washed with PBS (Invitrogen, Carlsbad, Ca). Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, Ca). Ganglia were homogenized in 600 µl lysis buffer. The samples were then processed following the manufacturer’s instructions. In the final step, the RNA was eluted with 50 µl of RNase-free water. This RNA isolation process was repeated four times for each group of 15 ganglia. First-strand cDNA synthesis was performed using random primers (Invitrogen, Carlsbad, CA). 1 µg of total RNA was denatured at 95 °C and reverse transcribed at 40 °C for 1 h using Superscript III Rnase H-RT (Invitrogen, Carlsbad, CA). Ten microliters of the RT product was added to the PCR mix containing primers for the mu opiate receptor. Primers and probes specific for the mu-opiate receptor gene (MOR) were designed by the software Primer Express (Applied Biosystems, Foster City, CA). The forward primer was 5’- ATGCCAGTGCTCATCATTAC-3’ and the reverse primer sequence was 5’- GATCCTTCGAAGATTCCTGTCCT-3’. The Taqman probe was constructed with the 5’-reporter dye, 6- carboxyfluorescein (FAM), and a 3’-quencher dye, 6-carboxy-tetramethyl-rhodoamine (TAMRA). The probe sequence was 5’- CGCCTCAAGAGTGTCCGCATGCT-3’. The endogenous control gene, β-actin, was used to normalize the RT-PCR. The 2X universal master mix (Applied Biosystems) containing the PCR buffer, MgCl2, dNTP’s, and the thermal stable AmpliTaq Gold DNA polymerase was used in the PCR reactions. In addition, 200 µM reverse and forward primers, 100 µM Taqman probe, 3 µl of RT product and Rnase/DNase-free water was added to the master mix to a final volume of 50 µl. The PCR reaction mixture was transferred to a MicroAmp optical 96-well reaction plate and incubated at 95 °C for 10 min to activate the Amplitaq Gold DNA polymerase and then run for 40 cycles at 95 °C for 30 s and 60 °C for 1 min on the Applied Biosystems GeneAmp 7500 Sequence Detection System(SDS). Each PCR was performed in triplicate. The PCR results were analyzed with the GeneAmp 7500 SDS 142 Month Jan FebMarchApril May June July Aug Sept Oct Nov Dec R el at iv e G en e E xp re ss io n 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Fig. 2 Relative mu opiate receptor gene expression in Mytilus edulis pedal ganglia determined by real time PCR for each monthly sample (n = 4). Paired t-tests revealed statistically significant differences between February or March and May or June or July (p = 0.004). software (Applied Biosystems). Relative gene expression was calculated using the method of Yoshikawa et al. (2001). Standard curves were generated by serial dilution of the June cDNA sample. R values were calculated and used to directly compare the monthly measurements. A paired student’s t-test was performed to evaluate differences between selected months. Results Morphine induced NO release measured on a monthly basis for one year revealed a average peak value of 39 ± 4 nM, during the late spring and early summer (Fig. 1). The lowest NO release occurred during the months of January (6.0 ± 0.5nM) through March (6.5 ± 1.1nM) (Fig. 1). The lowest sea surface temperatures (1.3 °C) were also recorded in these same three months (Fig. 1). Student’s t-tests revealed a statistically significant difference (p = 0.002) between the warm season high NO values and cold season low NO values. Relative gene expression was assessed by real time PCR. The mRNA expression reached a relative peak during the month of June and was at its lowest in February and March (Fig. 2). Student’s t-tests revealed a statistically significant difference (p = 0.004) between the warm season high R values (1.2 ± 0.10) and cold season low R values (0.46 ± 0.052). A regression analysis using mu opiate receptor expression as the independent variable and NO release as the dependent variable showed a correlation between the measurements. The calculated R value was 0.729. Discussion M. edulis neural tissues contain the typical biogenic amines, which includes dopamine (Stefano et al., 1976). Biogenic amines display variations in their ganglionic levels, which corresponds to the seasons and temperature, being high in warmer months and low in the winter months (Stefano et al., 1977a, 1977b; Stefano and Catapane, 1977a, 1977b). Interestingly, this same relationship occurs with opioid peptide expression along with their receptors (Stefano et al., 1980; Stefano and Leung, 1986). As demonstrated in this report, this same phenomenon involves NO release, which is coupled to opiate receptor activation, namely via μ3 ( Liu and Stefano, 1996; Liu et al., 1996; Magazine et al., 1996; Stefano and Scharrer, 1996; Stefano et al., 1996). The coupling of catecholamine, NO and morphinergic signaling has recently been reviewed (Kream et al., 2009; Stefano and Kream, 2009; Stefano et al., 2009; Zhu and Stefano, 2009). It is important to note that dopamine is a morphine precursor in this animal, which synthesizes endogenous morphine ( Zhu et al., 2005; Kream and Stefano, 2006). The significance of this precursor status of dopamine emanates from previously noted reports in this document showing seasonal and temperature alterations of catecholamine levels, which can now be directly compared to morphinergic phenomena, including the ability of morphine to release constitutively derived NO. We surmise that at colder “winter” temperatures all processes appear to be down regulated, including the homeostasis between an opiate receptor and its ligand levels, as currently demonstrated. This probably occurs because very cold temperatures influence all metabolic processes to decrease their activity levels, including those involved in various survival processes. This homeostasis mechanism occurs in all living organisms, including those with a pathogenic ability. Thus, in both types of organisms processes providing a survival benefit are not required. This 143 probably extends into the energy metabolic processes found in mitochondrial-like structures where morphine exerts actions (Kream and Stefano, 2009). In conclusion, the therapeutic value of performing medical operations, maintaining food, etc., at very low temperatures probably arises from the ability of low temperatures to disassociate adaptive cellular processes, allowing for a rather universal down regulation, which depending on the organism has tremendous survival advantage. In the case of Mytilus, if bacteria, viruses can’t survive or have a decreased infectious characteristic, why have a full functioning immune process with the activation of cytokines and opiate components at low temperature ( Stefano and Scharrer, 1994; Stefano and Salzet, 1999; Stefano et al., 2000, 2008; Stefano and Kream, 2008). References Cadet P, Mantione KJ, Stefano GB. 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