Dopamine, Morphine, and Nitric Oxide: An Evolutionary Signaling Triad

Abstract

Morphine biosynthesis in relatively simple and complex integrated animal systems has been demonstrated. Key enzymes in the biosynthetic pathway have also been identified, that is, CYP2D6 and COMT. Endogenous morphine appears to exert highly selective actions via novel mu opiate receptor subtypes, that is, mu3,‐4, which are coupled to constitutive nitric oxide release, exerting general yet specific down regulatory actions in various animal tissues. The pivotal role of dopamine as a chemical intermediate in the morphine biosynthetic pathway in plants establishes a functional basis for its expansion into an essential role as the progenitor catecholamine signaling molecule underlying neural and neuroendocrine transmission across diverse animal phyla. In invertebrate neural systems, dopamine serves as the preeminent catecholamine signaling molecule, with the emergence and limited utilization of norepinephrine in newly defined adaptational chemical circuits required by a rapidly expanding set of physiological demands, that is, motor and motivational networks. In vertebrates epinephrine, emerges as the major end of the catecholamine synthetic pathway consistent with a newly incorporated regulatory modification. Given the striking similarities between the enzymatic steps in the morphine biosynthetic pathway and those driving the evolutionary adaptation of catecholamine chemical species to accommodate an expansion of interactive but distinct signaling systems, it is our overall contention that the evolutionary emergence of catecholamine systems required conservation and selective “retrofit” of specific enzyme activities, that is, COMT, drawn from cellular morphine expression. Our compelling hypothesis promises to initiate the reexamination of clinical studies, adding new information and treatment modalities in biomedicine.

Introduction

It is apparent that evolutionary pressure has preserved and elaborated multifunctional chemical motifs commensurate with the exponential growth of complex biological systems. A prime example of evolutionarily driven combinatorial chemistry is the aromatic amino acid L‐tyrosine (L‐TYR). L‐tyrosine incorporates a critically important phenolic functional group as its chemical side chain. The biological significance of this key functional group is that hydroxylation of benzene to form phenol confers unique chemical advantages to L‐TYR and enhances its multifaceted utility in diverse physiological systems in both plants and animals.

Structurally, punctuation of polypeptide sequences with tyrosyl residues provides conformational integrity of cellular proteins by establishing hydrophilic interfaces with the aqueous environment. As a compelling corollary, key cellular developmental and regulatory processes are mediated by selective phosphate transfer to the phenolic hydroxyl group of protein bonded tyrosine residues by enzymes classified as tyrosine kinases.

Ongoing evolutionary modification of enzymes responsible for chemical modification of free L‐TYR has reinforced its preeminence as a precursor to major families of signaling molecules, most notably the catecholamines. Interestingly, the morphine biosynthetic enzyme pathway in opium poppy utilizes L‐TYR as its essential precursor molecule (Figure 1). Enzymatic modification of L‐TYR produces the required molecular building blocks required for morphine synthesis: tyramine, tyramine aldehyde, L‐3,4 dihydroxyphenylalanine (L‐DOPA), and dopamine (DA) [1].

Figure 1.

The initial steps in the biosynthesis of morphine leading to the formation of reticuline (reproduced from [1] with permission).

In opium poppy, morphine serves as a first‐line host defense molecule, termed phytoalexin, against microbial insult and the primary role of DA appears to be that of a biosynthetic intermediate [2]. Catecholamine signaling systems have evolved as mainstay regulators of integrated physiological and organ systems in animals [2]. Endogenous “morphinergic” signaling systems have evolved as autocrine/paracrine regulators of metabolic homeostasis, energy metabolism, and mitochondrial respiration. Catecholamine and endogenous “morphinergic” signaling systems share a common set of biosynthetic and metabolic enzymes, indicating significant evolutionary retrofitting of primordial enzyme species [1].

A second critical example of evolutionarily driven combinatorial chemistry is the basic amino acid L‐arginine (L‐ARG). The guanido group of L‐ARG is the primary carrier for free ammonia generated by protein and amino acid metabolism in the urea cycle of higher animals [3, 4, 5]. The importance of L‐ARG in maintaining systemic nitrogen balance is highlighted by the lethality of many inborn mutations of key enzymes of the urea cycle (Figure 2). Enzymatic oxidation of the guanido group of L‐ARG followed by selective cleavage and release of the free radical gas nitric oxide (NO) and the amino acid L‐citrulline has been preserved as a critically important cellular signaling mechanism throughout the course of evolution [6, 7, 8]. Evoked NO formation and release is mediated by a class of related Ca++‐dependent enzymes termed NO synthases (NOSs).

Figure 2.

Tyrosine and arginine end‐product interaction via selective stimulation of cNOS derived NO release. In general NO release from cNOS results in scavenging free radicals [79], antiviral and bacterial action [164], downregulation of immune, vascular, and neural tissue responsiveness as well as stabilizing micro environmental excitatory influences [36] besides functioning as a chemical messenger [73]. This ability of morphine to release NO exerting profound actions speaks to this long association. It is also important to note that the urea cycle produces arginine, representing the major nitric oxide source.

The crucial cellular metabolic and signaling roles of L‐TYR and L‐ARG are highlighted by the interactive regulatory activities of DA, morphine, and NO in diverse biological systems (Figure 2). These primordial regulatory relationships have been conserved to maintain an acceptable dynamic equilibrium between cellular levels of nitrogen and oxygen, which under certain physiological stressors become toxic. Evolutionary, developmental, and regulatory aspects of DA, “morphinergic,” and NO signaling systems are delineated below.

Evolutionary Persistence of Morphine and Emergence of Dopamine as a Signaling Molecule

In opium poppy, biosynthetic maturation and secretion of morphine and related benzylisoquinoline (BIQ) alkaloids is achieved by lactifer cells and serves as a first‐line host defense mechanism against microbial insult. Interestingly, the prototype catecholamine DA is exclusively utilized as an essential chemical substrate in the initial enzymatic Pictet‐Spengler condensation of DA and tyramine aldehyde to form norcoclaurine, the first committed chemical intermediate in the morphine biosynthetic pathway [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23] (Figure 1).

Evolutionary fate has preserved the ability to synthesize morphine throughout invertebrate phyla [24, 25, 26]. Recently, our group has demonstrated de novo biosynthesis of morphine from L‐TYR‐related aromatic molecules in animal cells [1, 27]. Endogenous morphine expression has also been demonstrated in human cancer cell lines [28]. In invertebrate tissues, catecholamine signaling has emerged with DA utilized as the preeminent signaling molecule found in neural and nonneural organ systems [29, 30, 31] and with limited involvement of norepinephrine (NE) [32] (Figure 3). Importantly, and despite numerous attempts, our laboratory could not identify epinephrine (E) in invertebrate tissues, suggesting that sequential hydroxylation and methylation of DA to form E had emerged at a relatively later stage of evolution. In vertebrates, it is established that the catecholamine pathway has been extensively elaborated and terminates in the synthesis and utilization of E [33].

Figure 3.

Illustration of morphine presence and significance in giving rise to the catecholamine pathway in vertebrates (DA, dopamine, NE norepinephrine, E epinephrine). In invertebrates and vertebrates, the catecholamine evolved into separate chemical messenger families.

We have previously advanced a hypothesis underlining the elaboration and evolutionary emergence of DA as a prototype signaling molecule linked to defined DA‐ergic/catecholaminergic signaling pathways apart from serving as a morphine precursor [2]. Taken together, both plants and animals make morphine and only in vertebrates is the catecholamine pathway full elaborated via highly regulated expression of E. These key relationships strongly suggest that catecholamine‐expressing signaling systems emerged from the morphine biosynthetic pathway via evolutionary adaptation of key enzymes involved in the modification of L‐TYR, L‐DOPA, DA, and tyramine [1, 2, 26, 27]. Morphine exerts general downregulation of tissue excitability via highly specific cellular and receptor mediated processes, protecting tissues from over excitability [34, 35, 36, 37]. With the advance of more complex motor activities in invertebrates and vertebrates associated with complex feeding, sexual, and protective processes a new signaling system had to emerge, namely, in the form of DA in invertebrates. Thus, DA may serve as a major signaling molecule associated with a mobile and motivated life style found in these animals [2].

It is our contention that this DA‐ergic regulatory function associated with mobility was so successful in invertebrates that it was continued in vertebrates with further amplification to fully establish NE and E signaling, further defining essential motor activation processes to better suit a more sophisticated mobile life style [2] (Figure 4). Thus, the initial motor activating system and its associated behaviors were amplified not only to include new complex motor activities but also highly developed motivation processes, that is, reward, pleasure, and pain, associated with the use of these systems were also expanded and developed [2] (Figure 4). Hence, we have the well‐recognized links between motor activities and emotional neural processes via common chemical messengers acting in a somewhat synergistic manner. We believe that he advent of the catecholaminergic signaling systems mediating complex emotive and cognitive processes emerged as a coping strategy serving as yet another means to activate motor processes in a more focused manner, providing for higher survival strategies [2, 38, 39].

Figure 4.

We surmise that early animals developed randomized motor activity first, which was then coupled to a system of reinforcing neural circuits that turned this activity to one that is guided, that is, motivational, linked to food and reproductory functions. This saved energy and made the motor activity purposeful. Further, the motivational component then developed an emotional component in higher animals that broke through the dangers of rationality, allowing decisions to be made by this cognitive “short‐cut”[39]. Interestingly, all components of the system are influenced by the same chemical messengers, demonstrating that it is integration that has changed during evolution.

Overlapping Chemical Substrates of Catecholaminergic and Morphinergic Expression in Animal Cells

Historically, anatomical detection of intrinsically low basal levels of immunoreactive morphine‐like material widely distributed across diverse central nervous system (CNS) areas may have led members of the scientific community to cursorily dismiss compelling arguments in support of a biological role for endogenous morphine expression [40, 41, 42, 43, 44, 45]. Because CNS distributions of immunoreactive morphine did not appear to be strictly colocalized with DA‐ergic systems, there appeared to be a conflict with prior data linking increased or aberrant production of DA metabolites to randomly formed DA‐derived BIQ alkaloids. Extensive data sets evolving from alcohol and Parkinson's disease research introduced inconclusive, often contradictory, evidence indicating that nonphysiological concentrations of BIQ alkaloids, often in the millimolar range, were required to mediate cellular toxicity via downregulation of necessary DA metabolism and turnover linked to free radical production. Because biologically meaningful concentration of BIQ alkaloids was often observed to have little or no effect on DA metabolism and cellular integrity, a null hypothesis was apparent indicating different, potentially important, regulatory activities for this class of biomolecules outside the realm of DA signaling.

In light of the above, the lack of a well‐characterized expression system made the difficulties of monitoring de novo incorporation of isotopically labeled L‐TYR, L‐DOPA, or DA into endogenous morphine appears insurmountable. Spector's group, however, made considerable advances in characterizing biosynthetic events involving in vivo enzymatic conversion of morphinan precursors into endogenous morphine, that is, the later stages of the biosynthetic pathway. Key studies demonstrated stereoselective conversion of the morphinan alkaloids (+)‐salutaridine, (–)‐thebaine, and (–)‐codeine into chemically authentic morphine in rat tissues [46] and transformation of thebaine to oripavine, codeine, and morphine by rat liver, kidney, and brain microsomes in the presence of NADPH‐ and NADH‐generating systems [47]. Importantly, use of chemical inhibitors indicated a critical role of cytochrome P450s (CYP) in these synthetic processes [47].

Contemporaneously, Goldstein and coworkers reported the presence of morphine‐like and codeine‐like immunoreactivities in bovine hypothalamus and adrenal, and in rat brain, that were subsequently analyzed as chemically authentic morphine and codeine [48, 49, 50]. Subsequently, the Goldstein group demonstrated in vivo and in vitro intramolecular conversion of reticuline to form salutaridine in rat liver, a critical step in generating the morphine/morphinan skeleton and the stereochemistry of the morphinan series [50]. Studies from Zenk and coworkers provided further characterization of hepatic conversion of reticuline to salutaridine [51, 52], thereby reinforcing the critical involvement of CYPs in the biosynthesis of endogenous morphine.

Neurochemical analysis of human brain tissue has detected and quantified very low levels of the biologically relevant (S) enantiomer of tetrahydropapaveroline (THP) that is selectively converted to (S)‐reticuline in the morphine biosynthesic pathway [53]. The absence of racemic THP also dispels serious consideration of the hypothesis that this essential morphine precursor is formed via random nonenzymatic Pictet‐Spengler condensation reactions of DA with 3,4‐dihydroxyphenylacetaldehyde [54, 55, 56, 57]. Similar considerations apply to the in vivo expression of nonracemic (R)‐salsolinol formed enzymatically from DA and acetaldehyde in extracted human brain samples [58, 59]. Demonstration of stereoselective expression of BIQ alkaloid precursors is complemented by later studies demonstrating the exclusive expression of the (S) enantiomer of the BIQ alkaloid morphine precursor (S)‐reticuline in cultured SH‐SY5Y human neuroblastoma and DAN‐G human pancreatic carcinoma cells [60, 61]. In these same analyses, ring‐labeled (S)‐THP, not (R), was stereoselectively incorporated into endogenous morphine via intramolecular isomerization of (S)‐ to (R)‐reticuline and enzymatic conversion to (R)‐salutaridine. Recent work from our group has demonstrated an approximate 3‐fold enhancement of tissue concentrations of endogenous morphine following administration of THP to an ex vivo preparation of invertebrate ganglia [62]. The observed rate of conversion of THP to morphine of approximately 20% when compared to the low steady‐state levels of tissue THP suggests a relatively high rate of conversion of THP and other morphine precursors including (R)‐reticuline and (R)‐salutaridine through a defined cellular biosynthetic pathway similar to that found in opium poppy.

Work from our group has demonstrated that polymorphonuclear neutrophil cells have the ability to synthesize morphine from small precursor molecules including L‐DOPA, reticuline, THP and tyramine, and DA in a concentration‐dependent manner [27]. Furthermore, additional studies utilizing the competitive CYP2D6 inhibitor bufuralol demonstrated a coordinate reduction in cellular morphine concentrations, thereby reinforcing the importance of CYP2D6 in these processes [1, 26, 27, 34, 63, 64]. More recent studies from our laboratory have demonstrated that substances of abuse (ethanol, cocaine, and nicotine) appear to have the ability to release endogenous morphine from various cells shown with compensatory increases in biosynthetic activity [65, 66, 67, 68].

In light of the above, we have hypothesized that the emergence of catecholaminergic signaling systems was facilitated by the genetic “retrofit” of a common set of enzymes within the morphine biosynthetic pathway to accommodate biochemical maturation and modification of DA‐related catecholamines [1, 2]. Notably, the plant N‐methyl and O‐methyl transferases required for conversion of the essential morphine precursor THP to the pre‐morphinan alkaloid S‐reticuline have been adaptively transformed into major enzymes in catecholamine expression, that is, phenylethanolamine N‐methyl transferase (PNMT) and catechol O‐methyl transference (COMT), respectively. Accordingly, evolutionarily driven chemical modifications of DA necessary for the cellular expression and utilization of E as a neural/neuroendocrine signaling molecule required coordinate recruitment and complex regulation of PNMT within tyrosine hydroxylase (TH)‐ and dopamine beta hydroxylase (DBH)‐positive cells [32]. Finally, it is likely that TH preceded DBH in the evolutionary scheme, reflecting the appearance of NE in select long lived invertebrates that required a higher level of motor associated mobilization strategies [32].

Intracellular Signaling Processes

We have suggested that evolutionary modification of enzymatic players within the morphine biosynthetic pathway was required to accommodate the establishment and chemical elaboration of catecholamine signaling pathways. The incorporation of NO‐mediated cellular signaling within morphinergic/catecholaminergic regulatory processes appears critical because of the uniform presence of NO across invertebrate and vertebrate phyla [36, 69, 70, 71] (Figure 2). Although TH and constitutive NO synthase (cNOS) share a common cofactor, that is, tetrahydrobiopterin (BH₄), the biological role of BH₄ in NO production is to serve as an accessory O₂ carrier [72, 73, 74, 75, 76]. Accumulated evidence indicates a critical cellular concentration of BH₄ is required for coupled electron transfer within the active site of cNOS. When electron transfer is uncoupled from L‐ARG oxidation the reactive oxygen species superoxide is formed. Increased superoxide production is associated with lowered NO production and is linked to increases in peroxynitrite concentrations and enhanced cellular oxidative stress [36, 77, 78, 79, 80, 81]. Furthermore, GTP cyclohydrolase (GCH) has been established as the rate‐limiting enzyme for BH₄ synthesis in animals, and thus has been shown to be a key modulator of peripheral neuropathic and inflammatory pain, which is also linked to NO signaling [71, 82].

Finally, it has been demonstrated that NO has significant and positive effects on cell survival that are antiapoptotic in DA‐positive neurons [83]. Recent critical studies have made the association between NO produced by a specific isotype found in the mitochondrion, that is, mitochondrial NOS (mtNOS), in regulating cellular oxygen consumption/energy metabolism without engendering oxidative stress [3, 84]. Taken together, our recent elucidation of a de novo morphine biosynthetic pathway in animal cells with strikingly similar characteristics to that found in opium poppy and morphine's ability to stimulate NOS derived NO release strongly suggests evolutionary pressure has conserved primordial regulatory circuitry [1]. It also suggests that this system has been carried over from a plant–animal common ancestor and manifests itself in energy and developmental processes [85].

Endogenous Morphine and Its Cognate 6 Transmembrane Helical Domain GPCRs

Our group has published biochemical, molecular, and pharmacological evidence for two unique 6 transmembrane helical domain (TMH) domain opiate receptors expressed from the μ opioid receptor (MOR) gene [86, 87, 88] (Figure 5). Designated μ3 and μ4 receptors, both protein species are Class A rhodopsin‐like members of the superfamily of G‐protein coupled receptors but are selectively tailored to mediate the cellular regulatory effects of endogenous morphine and related morphinan alkaloids via stimulation of NO production and release [35, 36, 37, 69, 87, 89]. Both μ3 and μ4 receptors lack an amino acid sequence of approximately 90 amino acids that constitute the extracellular N‐terminal and TMH1 domains and part of the first intracellular loop of the μ1 receptor, but retain the empirically defined ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains of the μ1 sequence. The binding profile of the μ3 and μ4 receptors is restricted to rigid BIQ alkaloids typified by morphine and its extended family of chemical congeners, it is hypothesized that conformational stabilization provided by interaction of extended extracellular N‐terminal protein domains and the extracellular loops is required for binding of endogenous opioid peptides as well as synthetic flexible opiate alkaloids.

Figure 5.

Schematic representation of 6 TMH μ opiate receptors. TMH domains of μ3 and μ4 receptors are numbered I–VI and correspond to conserved TMH domains 2–7 of the μ1 receptor. Conserved ELs, ILs, and C‐terminal intracellular sequences common to μ1, μ3, and μ4 receptors are represented by thick grey lines. Unique C‐terminal intracellular domains of μ3 and μ4 receptors are represented by the single‐letter amino acid code. The conserved intracellular N‐terminus of μ3 and μ4 receptors expressed from Exon 1 of the μ receptor gene is represented by the single‐letter amino acid code.

The evolutionary and biological significance of the μ3 opiate receptor is further enhanced by our recent finding that it is present on human stem cells as is its variant, the μ4 opiate receptor, in the absence of the traditional 7 TMH domain μ1 opioid receptor [89]. The ability of the μ3 opiate receptor subtype to gate intracellular calcium transients, eventually affecting mitochondrial oxygen consumption and energy conservation, provides a compelling functional linkage of this receptor with recently characterized calcium channels [90]. In effect, these evolutionarily conserved signaling and regulatory processes are coupled and functionally linked. It is also likely that the 6 TMH domain μ3 and μ4 opiate receptors were probably the prototypes in the line of future opioid receptor types that evolved in animals.

Endogenous Morphine, Dopamine, and Motivational Processes

With the advent of the μ3 opiate receptor and its coupling to constitutive NO release came the mechanism to “manage” evolving catecholaminergic signaling, which was needed to effectively control catecholamine activation‐type processes (feeding, movement, motivation, sex)? Thus, out of the “calming” homeostatic processes later emerged a system involved with selective activation (e.g., catecholamine), which would then be down regulated when their goal is accomplished.

The biological mechanism responsible for complex behaviors motivated by events commonly associated with pleasure is called “reward.” Reward‐directed behavioral processes are primarily mediated by limbic structures [39, 91, 92, 93, 94, 95, 96, 97, 98, 99] and are heavily dependent on DA‐ergic signaling [92]. Recent findings have drawn an association of endogenously expressed morpine with reward‐directed behavioral processes [24, 37, 43, 100]. Additionally, other neurotransmitters (e.g., GABA, glutamate, serotonin, stress hormones) may play a critical role [101, 102, 103]. Naturally rewarding experiences including feeding and sex in accordance with other substances, such as caffeine, ethanol, nicotine, cocaine, and alcohol may also activate the brain's reward and motivation circuitries [67, 91, 103, 104]. The most powerful drugs of abuse associated with addictive reward processes include the psychomotor stimulants (e.g., amphetamine, cocaine) and opiates (e.g., heroin, morphine). Thus, the ability of addictive drugs to strongly activate CNS reward systems and to chemically alter normal functions of these systems is a crucial feature of addiction/substance abuse [34, 91, 105, 106, 107, 108]. State‐dependent alterations in reward circuitry acquired during the course of pleasure‐seeking behavior and drug abuse potentially promote tolerance, dependence, craving, relapse, and vulnerability [92, 99, 109].

It has been established that drugs of abuse mediate evoked release of DA from the ventral tegmental area (VTA) into the nucleus accumbens, thereby altering responsiveness to glutamate within the prefrontal cortex [92, 98, 99]. Changes in sensitivity to glutamate may then enhance both the release of DA from the VTA and responsiveness to DA in the nucleus accumbens, thereby promoting CREB and delta FosB activity [92, 109]. Yet, with prolonged abstinence, changes in delta FosB activity and glutamate signaling predominate [98, 99, 109, 110, 111]. These actions may trigger relapse by increasing sensitivity to the drug's effects (if used again), eliciting powerful responses to memories of past highs and cues that bring those memories to mind [92, 98, 112]. Counter intuitively, abstinence from cocaine or morphine after repeated administration may also decrease DA levels in the mesolimbic DA system/VTA [113, 114]. Compromised DA‐ergic signaling may be related to the intense craving associated with withdrawal in human drug addicts [91].

Various addictive drugs share the common feature of stimulating the same DA‐ergic brain reward system (e.g., heroin enhances DA levels by increasing DA release, whereas cocaine inhibits DA reuptake), and this action has been related to their appetitive motivational effects [91, 92, 115]. Opiates may further generate a strong “reward message” by acting directly on the nucleus accumbens [92, 116]. Again, we find the complexity that operationally directs DA‐ergic reward processes.

Recent work from our group has solidified functional linkages between DA‐ergic and “morphinergic” signaling pathways [1, 26, 27]. Importantly, DA serves as a major precursor involved in the cellular expression of endogenous morphine [1, 26, 27]. Therefore, aberrations in DA metabolism may induce modifications in morphine synthesis and consequently weaken endogenous morphine's action on the “DA” reward system [24, 103, 117].

The ramifications of multifaceted DA‐ergic and morphinergic regulatory processes are realized in complex behavioral endpoints. From this perspective, it is seen that hyperactivity, aggression, and rage are first manifested followed by a period of low‐level activity somewhat like relaxation [2]. The biphasic manifestation of hyperactivity followed by relaxation has significant evolutionary value. It would be advantageous for the organism to always be in a state of alertness that is regulated by DA‐ergic processes [118, 119, 120]. This would allow the organism to be ready for an unexpected event that may be life‐threatening. Once an organism considers this type of threat nonexistent or whose probability is diminished the relaxation mediated by morphinergic processes may emerge at this appropriate time and allow for relaxation and reward activities. The combination of events may arise from different manifestations of functionally integrated morphinergic and DA‐ergic signaling processes.

Dysregulation of Catecholaminergic and Morphinergic Regulatory Processes in Pathophysiological States

Initial speculations as to the existence and potential physiological role of endogenous morphine were made over 30 years ago by prominent researchers in the field of alcohol abuse, not opiate abuse, who advanced the hypothesis that the reinforcing or addictive effects of ethanol were functionally linked to the cellular effects of DA‐derived isoquinoline alkaloids, notably the tetrahydroisoquinoline (TIQ) salsolinol [54, 55, 121] and the BIQ morphine precursor THP [122, 123, 124]. Recognition of TIQs, THP, and endogenous morphine as active principles of alcohol abuse was inherently linked to their normal presence in DA‐positive neurons, enhanced cellular expression following chronic ethanol intake [56, 62, 65, 124, 125, 126], and concentration‐dependent dysregulation of DA metabolism and/or DA‐ergic signaling in mesocortical/mesolimibic areas such as the nucleus accumbens (NAC) and VTA traditionally associated with reinforcement of alcohol‐related behaviors [127, 128, 129, 130, 131]. The causal relationship and functional association of CNS expression of TIQ and BIQ alkaloids to alcohol abuse remains controversial despite anatomical, physiological, pharmacological, and behavioral evidence linking DA‐ergic and opioidergic systems in limbic areas associated with reinforcement of ethanol intake behaviors [58, 132, 133, 134].

The functional association between aberrant DA metabolism, cellular expression of isoquinoline alkaloids, and the etiology of Parkinson's disease has also been extensively studied and debated for three decades [57, 59, 135, 136, 137, 138, 139, 140, 141]. In contrast to the hypothesized role of isoquinoline alkaloids to activate neural circuits involved in the reinforcement of alcohol dependence, these same conjugate molecules were proposed as pathophysiological agents responsible for Parkinson's disease‐associated symptomatology. Interestingly, by the early 1970s a functional association between L‐DOPA therapy and in vivo formation of BIQs had been proposed [142, 143, 144, 145]. It was subsequently demonstrated that urinary levels of morphine, codeine, and THP in L‐DOPA‐treated Parkinsonian patients are dramatically elevated as compared to matched controls and abstinent alcoholics [146]. Not surprisingly, enhanced production of THP in Parkinsonian patients was peremptorily linked to the mediation of adverse side effects and cellular toxicity evolving from chronic L‐DOPA therapy [147, 148, 149, 150, 151, 152], despite clinical evidence supporting positive effects of morphine on L‐DOPA‐associated dyskinesias [153, 154, 155, 156].

Operationally, it is apparent that the morphine biosynthetic pathway may be found entirely within a single neuron whereby DA may or may not be used as a signal molecule, and simply serve in the capacity of morphine precursor. Even within this scenario, it may be possible for the DA to serve as a signal molecule appearing first subsiding, leading to the release or use of morphine as the last signal molecule. It may also be possible that if this was a pure morphine neuron that DA associated with it may come from a pure DA neuron not making morphine. In this scenario, once DA is released it could then be transported into a morphine neuron whereby it enters into morphine biosynthesis. In either scenario, morphine biosynthesis occurs within this tissue.

From behavioral perspectives, DA‐ergic signaling systems mediate behavioral processes that underlie hyperactivity, excitability, rage, and aggression, whereas “morphinergic” signaling modulates the downregulation of these states, that is, generation of a state of calm relaxation. Thus, one may surmise that in depression the DA‐ergic system may be heavily down regulated by morphine signaling. In schizophrenia, this relationship may manifest itself in an on/off dysregulation via an apparent morphine signaling process thereby allowing for altered hyperactivity states. Alternatively, aberrant DA‐ergic signaling may not be able to be suppressed by a normally functioning morphinergic signaling system. This type of abnormality may represent an underpinning for substance abuse or the potential for substance abuse. As noted earlier it was interesting to discover that cocaine, nicotine, and alcohol all have the ability to release endogenous morphine from normal healthy cells. This information certainly goes a long way in explaining how interactive DA‐ergic/morphinergic regulatory loops function under normal circumstances as well.

It comes as no surprise to comprehend what happens when variations/errors creep into the modulation of the activation processes. Furthermore, the catecholamine emergence from the older morphinergic biosynthesis pathway provides a compelling rationale for all the literature coupling of these and other ancillary pathways into morphinergic signaling processes, providing the missing knowledge for various disciplines such as substance abuse as well as mental health since both depend on a catecholamine signaling substrate.

Conclusions

In conclusion, ongoing research to elucidate reciprocal regulatory roles of DA‐ergic, morphinergic, and NO‐ergic signaling pathways will establish a compelling translational platform by which to evaluate and treat a variety of pathophysiological states. Polymorphisms in human genes expressing key enzyme activities including GCH and COMT provide naturally occurring models for determining the individual contributions of DA‐ergic, morphinergic, and NO‐ergic signaling pathways to concerted homeopathic regulation of metabolic activity in cell and organ systems as discussed elsewhere [103, 118, 157, 158]. The established status of DA as an essential intermediate in the expression of endogenous morphine biosynthesis lends considerable insight for understanding the biochemical and molecular bases of complex excitatory and inhibitory behaviors. Accordingly, the autocrine/paracrine nature of DA‐ergic, morphinergic, and NO‐ergic signaling processes is predicted to undergo rapid regulation via feedback/end product inhibition by individual chemical messengers. We have established NO as the final chemical messenger appropriately expressed within this regulatory pathway, thereby designating cGMP activation as a potential key player in the profound downregulatory activities of endogenous morphine [36, 159, 160, 161]. Furthermore, enhanced NO production has been demonstrated to mediate inhibitory effects on gene expression of several enzyme activities required for endogenous morphine biosynthesis [162, 163]. Taken together, ongoing elucidation of critical aspects of this novel triadic signaling relationship promises to resolve as well as create novel opportunities for gaining insights into normal function, as well as those driving pathophysiological disorders including cardiovascular disease, psychiatric illnesses, pain, and addiction [71].

Conflict of Interest

The authors declare no conflict of interest.