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. 2006 Apr 28;26(4-6):1035–1043. doi: 10.1007/s10571-006-9021-4

Norlaudanosoline and Nicotine Increase Endogenous Ganglionic Morphine Levels: Nicotine Addiction

Wei Zhu 1, Kirk J Mantione 1, Lihua Shen 1, Brian Lee 1, George B Stefano 1,✉
PMCID: PMC11520596  PMID: 16645895

Abstract

1. Given the presence of morphine, its metabolites and precursors, e.g., norlaudanosoline, in mammalian and invertebrate tissues, it became important to determine if exposing normal excised ganglia to norlaudanosoline would result in increasing endogenous morphine levels.

2. Mytilus edulis pedal ganglia contain 2.2 ± 0.41 ng/g wet weight morphine as determined by high pressure liquid chromatography coupled to electrochemical detection and radioimmunoassay.

3. Incubation of M. edulis pedal ganglia with norlaudanosoline, a morphine precursor, resulted in a concentration- and time-dependent statistical increase in endogenous morphine levels (6.9 ± 1.24 ng/g).

4. Injection of animals with nicotine also increased endogenous morphine levels in a manner that was antagonized by atropine, suggesting that nicotine addiction may be related to altering endogenous morphine levels in mammals.

5. We surmise that norlaudanosoline is being converted to morphine, demonstrating that invertebrate neural tissue can synthesize morphine.

KEY WORDS: morphine, norlaudanosoline (tetrahydropapaveroline), nervous tissue, invertebrates, ganglia, endogenous morphine, nicotine, atropine, acetylcholine

INTRODUCTION

Over the last 30 years, evidence is accumulating that supports the endogenous morphine signaling hypothesis in animals. There is a body of evidence demonstrating that opiate alkaloids, such as morphine, morphine-3- and 6-glucuronide, as well as the morphine putative precursor molecules (thebaine, salutaridine, norcocolarine, reticuline and codeine), exist in vertebrates (Donnerer et al., 1986; Lee and Spector, 1991; Epple et al., 1994; Zhu et al., 2003) and in the neural tissues of the marine bivalve Mytilus edulis (Stefano et al., 1993, 2000b; Goumon et al., 2001; Zhu et al., 2001a, 2002b). Taken together, this data has suggested that animals can make morphine.

In recent years, we have demonstrated that exposing M. edulis pedal ganglia to reticuline increases endogenous ganglionic morphine levels (Zhu and Stefano, 2004). L-DOPA injection into normal and healthy M. edulis has also resulted in an increase in ganglionic morphine levels (Zhu et al., 2005c). In addition, these animals contain norlaudanosoline (tetrahydropapoverine), another precursor in the proposed animal morphine biosynthetic pathway (Zhu et al., 2002b). These data provide important evidence, supporting the hypothesis, that animals have the ability to synthesize morphine.

In the present report, we demonstrate that norlaudanosoline also has the ability to augment endogenous ganglionic morphine levels in vitro, demonstrating that morphine synthesis can occur in this animal in a manner similar to that found in plants (Brochmann-Hanssen, 1985; Stefano and Scharrer, 1994). Furthermore, we demonstrate, because this animal also exhibits cholinergic signaling, that nicotine in a receptor mediated manner also can enhance endogenous morphine levels. Thus, both precursor addition and pharmacological alteration supports the hypothesis that morphine is an endogenous signaling molecule.

MATERIAL AND METHODS

M. edulis collected from the local waters of Long Island Sound were maintained as previously described in detail (Stefano et al., 1994). For the biochemical analysis, groups of 20 animals had their pedal ganglia excised at different time periods and incubated with different concentrations of norlaudanosoline, ranging from 1 to 100 ng/mL. In separate experiments, different animals were injected with cholinergic agents, e.g., nicotine and atropine, at the indicated concentrations. After 1 h, their pedal ganglia were excised on ice, pooled (15 per treatment) and assayed for morphine as described below.

Morphine Determination, Solid Phase Extraction

The morphine extraction protocol was performed in pooled ganglia as reported in detail elsewhere (Zhu et al., 2001a,b, 2002b, 2003; Zhu and Stefano, 2004). The dried extract was then dissolved in 0.05% trifluoroacetic acid (TFA) water before solid phase extraction. Samples were loaded on a Waters Sep-Pak Plus C-18 cartridge previously activated with 100% acetonitrile and washed with 0.05% TFA-water. Morphine elution was performed with a 10% acetonitrile solution (water/acetonitrile/TFA, 89.5%:10%:0.05%, v/v/v). The eluted sample was dried with a Centrivap Console and dissolved in water prior to high pressure liquid chromatography (HPLC) analysis.

Radioimmuno-Assay (RIA) Determination

The morphine RIA determination is a solid phase, quantitative RIA, wherein125I-labeled morphine competes for a fixed time with morphine in the test sample for the antibody binding site. The commercial kit employed is from Diagnostic Products Corporation (Los Angeles, CA) (Zhu et al., 2001a,b, 2002b, 2003; Zhu and Stefano, 2004). The detection limit was 0.5 ng/ml.

HPLC and Electrochemical Detection of Morphine in the Sample

The HPLC analyses were performed with a Waters 626 pump (Waters, Milford, MA) and a C-18 Unijet microbore column (BAS). A flow splitter (BAS) was used to provide the low volumetric flow-rates required for the microbore column. The split ratio was 1:9. Operating the pump at 0.5 ml/min, yielded a microbore column flow-rate of 50 μl/min. The injection volume was 5 μl. Morphine detection was performed with an amperometric detector LC-4C (BAS, West Lafayette, Indiana). The microbore column was coupled directly to the detector cell to minimize the dead volume. The electrochemical detection system used a glassy carbon-working electrode (3 mm) and a 0.02 Hz filter (500 mV; range 10 nA). The cell volume was reduced by a 16-μm gasket. The chromatographic system was controlled by Waters Millennium32 Chromatography Manager V3.2 software and the chromatograms were integrated with Chromatograph software (Waters).

Morphine was quantified in the tissues by the method described by Zhu et al. (2001a). Several HPLC purifications were performed between each sample to prevent residual morphine from remaining on the column. Furthermore, mantle tissue was run as a negative control, demonstrating a lack of contamination (Zhu et al., 2003). All solutions devoid of animals or their tissues were also examined for any detectable morphine and none was found.

RESULTS

M. edulis pedal ganglia extracts contain morphine, confirming earlier studies that also identified this material via Q-TOF mass spectrometry (Zhu et al., 2001a). Incubation of the ganglia in vitro with various concentrations of norlaudanosoline increases ganglionic morphine levels after one hour in a concentration and time dependent manner (Fig. 1A and B; P < 0.01). The increase in ganglionic morphine levels, after norlaudanosoline exposure, occurs gradually over the 60 min incubation period (Fig. 1). We estimate that approximately 10% of norlaudanosoline gets converted to morphine. Blank runs between morphine HPLC determinations, as well as running a negative tissue control, did not show a morphine residue with RIA. Analysis of the marine water and various chemicals used in the protocol also demonstrated a lack of morphine. Incubation of ganglia with 10−6 M tryptophan did not alter basal morphine levels after 1 h (data not shown).

Fig. 1.

Fig. 1.

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Norlaudanosoline enhances endogenous ganglionic morphine levels in Mytilus edulis pedal ganglia. (A) Ganglia (20 per treatment) were incubated with 1.0, 10, 50, and 100 ng of norlaudanosoline for 60 min. Morphine quantification was by RIA. One Way ANOVA analysis shows the morphine level in ganglion incubated with norlaudanosoline were significantly higher (*** denotes P < 0.001) than control at 50 and 100 ng of norlaudanosoline (THP). (B) Norlaudanosoline (100 ng/ganglia) incubation of Mytilus edulis pedal ganglia exhibit an increase in endogenous morphine levels, which is time-dependent. Saline incubation served as a control. Morphine levels were determined RIA. One Way ANOVA analysis shows the morphine level in ganglia incubated with norlaudanosoline was significantly higher than control at 30 and 60 min (*** denotes P < 0.001). All determinations were replicated four times and the mean graphed ± SEM.

Injection of nicotine (10 μg/animal under the foot) in a 100 μl volume significantly increased ganglionic morphine levels in a manner that was blocked by concomitant atropine (10 μg in a 100 μl volume) and nicotine exposure (Fig. 2). Atropine alone had no influence on ganglionic morphine levels.

Fig. 2.

Fig. 2.

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Cholinergic influences on ganglionic morphine levels. Animals were injected in the foot with nicotine (10 μg/100 μl), atropine (10 μg/100 μl) or a mixture of both chemicals for 1 h. Each group contained 15 animals. Ganglia were dissected and extracted (see Section ‘‘MATERIALSAND METHODS’’). One way ANOVA analysis shows the morphine level in the ganglia injected with nicotine were significantly higher (*** denotes P < 0.001) than control (saline injection). Atropine alone did not affect morphine level in the ganglia; also, injection of atropine with nicotine did not increase morphine level. Morphine levels were determined by RIA. All treatments were replicated three times and mean graphed ± SEM.

DISCUSSION

The present study demonstrates that exposing pedal ganglia to the putative morphine precursor, norlaudanosoline (Stefano and Scharrer, 1994; Zhu et al., 2003), results in significant increases in ganglionic morphine levels, which is time- and concentration-dependent. Additionally, we surmise that ganglionic acetylcholine may have the ability to modulate ganglionic morphine synthesis since nicotine also enhanced its levels via a receptor-mediated mechanism. It is apparent that M. edulis neural tissues have the ability to synthesize morphine from norlaudanosoline.

Morphine precursors have been identified in mammalian and invertebrate tissues (Goldstein et al., 1985; Donnerer et al., 1986; Kodaira and Spector, 1988; Kodaira et al., 1989; Goumon and Stefano 2000), including parasites (Stefano et al., 1993, 2000b; Leung et al., 1995; Sonetti et al., 1999; Goumon et al., 2000, 2001; Zhu et al., 2001a, 2002a, 2004b). The first report of an animal making morphine was in the parasite Ascaris suum (Goumon et al., 2000). Further supporting the endogenous presence of morphine in animals is the cloning of the mu3 opiate receptor subtype found in human and invertebrate tissues, which only responds to opiate alkaloids, i.e., morphine, as opposed to opioid peptides and provides the means for endogenous morphine signaling (Stefano et al., 1993; Cadet et al., 2003) (Fig. 3).

Fig. 3.

Fig. 3.

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Morphine formation in M. edulis pedal ganglia (see Zhu et al., 2005b). Our previous and present report demonstrates that morphine is synthesized by the precursors listed in the fig. CYP2D6, a member of CYP450 enzyme family, mediates the conversion of tyramine to dopamine, reticuline (or norlaudanosoline) to morphine, and codeine to morphine. Other enzymes also are involved and will be the subject of future work.

The argument for a de novo biosynthetic pathway in animals can also be supported with studies demonstrating the ability of animal enzymes to synthesize, through the same precursors, morphine in an identical stereo- and regio-specific manner to that of the poppy plant (Yamano et al., 1985; Kodaira et al., 1989; Amann and Zenk, 1991; Sindrup et al., 1993; Amann et al., 1995; Zhu et al., 2003). This observation was recently substantiated by Zenk and colleagues, demonstrating that labeled tyrosine can be made into morphine in vitro in a human cancer cell line (Poeaknapo et al., 2004).

Recently, in normal and healthy M. edulis pedal ganglia and in vivo, we demonstrated that this animal can synthesize morphine from tyrosine via dopamine in a process mediated, in part, by CYP2D6, a cytochrome P450 isoform (Zhu et al., 2005b). This same process has also been demonstrated in human white blood cells taken from healthy donors (Zhu et al., 2005a). These reports demonstrate, by precursor incubation, enzyme inhibition and nucleic acid sequencing, that diverse animals have the ability to synthesize morphine.

Tetrahydroisoquinoline alkaloids, like norlaudanosoline, belong to a group of naturally occurring pharmacologically active compounds. They are taken up, stored, and released from nerve terminals, and interfere with normal catecholaminergic transport and storage systems (Heikkila et al., 1971; Greenberg and Cohen, 1973). Norlaudanosoline is an endogenous 1-benzyltetrahydroisoquinoline previously found in both plant and animal tissues, including human (Turner et al., 1974; Brochmann-Hanssen, 1985; Zhu et al., 2002b). It is synthesized by a non-enzymatic Pictet-Spengler reaction of dopamine with 3,4-dihydroxyphenyl-acetaldehyde (Davis and Walsh, 1970; Greenberg and Cohen, 1973).

Moreover, it appears that animals synthesize morphine in a manner similar to that of the poppy plant (Zhu et al., 2003, 2005a,b; Neri et al., 2004). Reticuline, norlaudanosoline, -DOPA, tyrosine and tyramine all appear to be part of the animal morphine biosynthetic pathway (Stefano and Scharrer, 1994; Stefano et al., 2000b; Zhu et al., 2003, 2005c; Poeaknapo et al., 2004; Sonetti et al., 2005; Casares et al., 2005).

We have surmised that endogenous morphine, in general, is involved with down regulating various physiological activities (Stefano and Scharrer, 1994; Stefano, 1998; Stefano et al., 2000a,b, 2005a,b; Sonetti et al., 2005; Guarna et al., 2005; Mantione et al., 2005; Fricchione and Stefano, 2005). It is specifically this activity that may have led to its continuous presence in animals.

Acetylcholine is also present in mollusks as an endogenous signaling substance, along with muscarinic- and nicotinic-like receptors (Vehovszky and Salanki, 1983; Lee et al., 1998; Wright and Huddart, 2002; Dowell et al., 2003). This same situation exists in M. edulis (Hellwig and Achazi, 1991). In a somewhat parallel story with opiate alkaloid addiction (Esch and Stefano, 2004), nicotine also is addictive (Dani and Harris, 2005). As with morphine, it exerts profound cellular and synaptic actions that modulate motivational and behavioral changes that can be associated with addiction (Esch and Stefano, 2004; Zhu et al., 2004a; Guarna et al., 2005; Fricchione and Stefano, 2005; Pryor et al., 2005; Dani and Harris, 2005). As noted earlier, we have recently demonstrated that morphine can be made by invertebrate ganglionic tissues and human white blood cells in a process mediated, in part, by CYP2D6 (Zhu et al., 2005a,b). Again, this parallels the nicotine metabolic pathway in that it, too, is mediated by a CYP 450 isoform, namely CYP2A6 (Nakajima and Yokoi, 2005). Nicotine is C-oxidized to cotinine, which is catalyzed by CYP2A6 (Nakajima and Yokoi, 2005). Thus, it would appear that links may exist in these pathways, coupling these signaling molecules in reward and pain pathways, as well as others. Taken together, the results of the present study demonstrate that nicotine enhances ganglionic morphine levels, suggesting this as a role for ganglionic acetylcholine. It also strongly suggests nicotine's addictive properties may arise from this ability to enhance endogenous morphine levels, opening up a new level of understanding in nicotine induced addiction and behavioral effects as well as morphine regulation.

ACKNOWLEDGMENTS

This work was supported, in part, by the following grants: NIMH 47392. Mr. Brian Lee is a member of a High School Research Program in association with the Long Island Conservatory.

Abbreviations:

HPLC

High pressure liquid chromatography

THP

tetrahydropapoverine/norlaudanosoline

PBS

phosphate buffered saline

RIA

radioimmunoassay

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