English
中文ABSTRACT
The alkaloid compound morphine is one of the strongest known opioid analgesic compounds and has a wide range of therapeutic applications (Yaksh 1997). Morphine was first isolated from the opium poppy Papaver somniferum, where it can be found in high levels (Shukla et al 2006). Trace amounts of morphine have been found in human tissues and fluids, where it was believed to be dietary in origin, coming from the ingestion of morphine-containing plant substances. Recent findings have disputed this fact and have raised questions over whether the traces of morphine found in humans and animals are dietary or endogenously synthesised.
Endogenous morphine receptors (mu, delta, kappa) were discovered to be present in the human body, leading to the hypothesis that endogenously produced substances must therefore act at these receptors (Chang et al 1979). Further discovery of an endogenous mu receptor sub-type, mu3 receptor, selective for opiate alkaloids, strengthened the argument that humans can synthesise opiate alkaloids (Pasternak 2001). Endogenous morphine has been discovered in several human cell lines, including plasma and cancer cells, suggesting that human cells have the capability of synthesising morphine (Poeaknapo et al 2004).
Although the exact pathway for the biosynthesis of morphine is still not fully understood, it is thought to involve at least 19 steps (Boettcher et al 2005). The CYP2D6 isoenzyme has been identified as a possible enzyme utilised in the biosynthesis of morphine and may be involved at critical steps in this biosynthetic pathway (Zhu et al 2005).
INTRODUCTION
The pain relieving and euphoric effects of opium, extracted from the opium poppy, have been known for centuries. The major active constituent of opium is the alkaloid morphine (Evans 2004). Morphine is one of the strongest analgesic compounds known and much research has gone into exploring its properties (Rang et al 1998).
Until recent years, it was believed that morphine could not be synthesised by animals (Evans 2004). The trace amounts of morphine isolated from animals, for example human and cows’ milk, were believed to be dietary in origin, coming from the ingestion of morphine-containing plants (Hazum et al 1981). Endogenous morphine has now been successfully isolated and authenticated in trace amounts from human tissue using mass spectrometry and its origins have been subject to much research (Poeaknapo et al 2004).
What is endogenous morphine?
The discovery of multiple endogenous opioid receptors in animals and humans stimulated the search for naturally-occurring opioid substances (Martin 1979). Endogenous substances were discovered that acted as agonists to these opioid receptors. It therefore became possible to start the characterisation of these newly discovered endogenous opioids (Corbett 2006).
The first endogenous opioid receptor ligands to be discovered were [Met]- and [Leu]- enkephalin (Dhawan et al 1996). It was later discovered that a fragment of a pituitary hormone also contained the amino acid sequence of enkephalin. This substance was called β-endorphin and was shown to be a potent agonist for opioid receptors (Aunis D et al 2006)
These naturally-occurring opioid compounds, such as β -endorphin, demonstrated that there were indeed endogenous opioids of similar structure to morphine present in humans. Morphine itself binds strongly to these receptors which suggests there may also be a strong possibility that morphine is produced endogenously. Why would morphine have such a strong affinity for the human opioid receptor if it is not naturally synthesised by humans? Moreover, endogenous morphine has been found to be present in low concentration in several human tissues and fluids (Poeaknapo et al 2004).
DISCUSSION
Evidence for endogenous morphine synthesis
In order to demonstrate that opioid substances can be endogenously synthesised, the availability of substrates and a viable biosynthetic pathway needed to be identified.
Human plasma contains low but physiologically significant concentrations of morphine (Poeaknapo et al 2004). In a study by Zhu et al, it was found that human white blood cells, particularly polymorphonuclear cells, are able to synthesise morphine from tyramine, norlaudanosoline and codeine (Zhuet al 2005). This study used white blood cell cultures to investigate how the isoenzyme CYP2D6 may be implicated in the synthesis of morphine. In addition, the study elucidated an alternative pathway for morphine synthesis via L-DOPA. The researchers additionally showed that white blood cells secrete morphine into their environment and thus regulate themselves and other cells via mu3 receptors. This evidence goes to suggest that human immune cells have the capability to synthesise morphine.
In a study to investigate endogenous morphine production in animals, researchers showed that animals, namely molluscs, had the ability to synthesise morphine in a dynamic process using two different pathways, one involving L-tyrosine and the other involving L-DOPA (Cadet et al 2005 & Sonetti et al 2005). This alternative L-DOPA pathway was later confirmed using both in-vitro and in-vivo studies on human tissue (Zhu et al 2005). Importantly, the Cadet study discovered that the inhibition of one pathway allowed the other pathway to continue with morphine synthesis. This evidence suggests that the two morphine biosynthesis pathways are coupled, where inhibiting one can increase the activity of the other. The discovery of two pathways has significant biomedical significance as there are implications for the use of the alterative pathways in treatment of diseases such as Parkinson’s disease. Morphine levels seem to increase following L-DOPA exposure, and Parkinson’s disease is closely linked to dopamine concentrations in the brain (Zhu 2003).
The biosynthetic pathway for endogenous morphine
In a study by Boettcher et al, it was discovered that there are over 19 steps in the main biosynthetic pathway for morphine. Microsomal cytochrome P450 (CYP) isoenzymes are critically important in this biosynthesis of morphine (Boettcher et al 2005). CYP2D6 is an isoenzyme found on chromosome 22. It is expressed in neural, immune and hepatic tissues of animals and humans (Touw 1997). CYP2D6 is involved in the metabolism of many endogenous compounds such as steroids, amines and fatty acids. It is also involved in the oxidative metabolism of pharmaceutical compounds such as beta-blockers and anti psychotics (Flockhart 2006). CYP2D6 appears to act at critical steps in the morphine biosynthetic pathway in polymorphonuclear cells of rats (Bianchi GB, 2003) as well as in human tissues (Cho T, 2003). (See figure 1)
Figure 1: The involvement of CYP2D6 at various steps in the biosynthetic pathway of morphine. Top: Tyramine is hydroxylated to form dopamine. Middle: (R)-reticuline is metabolised through a p-ortho-oxidative coupling of (R)-reticuline, which is catalysed by microsomal cytochrome P450 enzyme CYP2D6. Bottom: The human CYP2D6 is involved in the demethylation of codeine to morphine.
Human neuroblastoma cells (SH-SY5Y) have the ability to synthesise morphine via two biosynthetic pathways (see Table 1) (Boettcher et al 2005). It is unclear whether it is possible to translate this tumour cell activity to normal cell biosynthesis, however, this is useful in studying the possible biosynthetic pathway for morphine applied to human cells. Human morphine biosynthesis is thought to involve at least 19 chemical steps which start via a metabolic route beginning with L-Tyrosine (Boettcher et al 2005). These steps seem to closely resemble the biosynthetic pathway found in the opium poppy. There is however a fundamental difference in the formation of a key intermediate, (s)-reticuline. The formation (s)-reticuline proceeds via (S)-norlaudnanosoline whilst plant morphine biosynthesis proceeds via (S)-nordoclaurine (Boettcher et al, 2005). This is an interesting discovery as it goes to suggest that humans possess their own unique pathway for synthesis of endogenous morphine. In order to demonstrate compelling evidence for the biosynthesis of morphine endogenously in human beings, it will be necessary to have more evidence of regulatory mechanisms in place for the biosynthetic process in cells. This should be applied to normal healthy human cells and tissue rather than cancer cells alone.
Another area of research being investigated is the utilisation of particular substrates in the morphine biosynthesis pathway. The exact mechanisms for the compartmentalisation and mobilisation of substrates for morphine synthesis, such as L-tyrosine and L-tyrosine-derived products, are needed. One possible mechanism that has been proposed for this mobilisation of substrates is the presence of reversible transamination of L-Tyrosine via pyruvic acid intermediates (Kream et al 2006). There is no doubt however that further studies will be required in this area to provide conclusive data before firm conclusions can be made concerning the substrates used in morphine biosynthesis.
The mu3 receptors
Once synthesised, the endogenous opioid compounds must then bind to a receptor site within the body. A novel opiate mu receptor mu3, a subtype of the mu1 receptor, has been discovered in studies involving cloned mu receptors (Bartlett et al 2004). This mu3, opioid receptor is expressed in human monocytes, granulocytes, vascular and endothelial cells, in addition to several other tissues (Pryor 2005). It is selective for opiate alkaloids such as morphine, again reinforcing the theory that morphine is an endogenous compound in humans (see figure 2) (Cadet et al 2003). On binding to the mu3 receptor, a signal transduction cascade is activated via G-protein coupling. The G-protein is a membrane protein that acts as a ‘gateway’ that allows a response from the cell. This measured response to opioid compounds suggests that there is a possible role for mu receptors and endogenous morphine in human haematopoietic stem cell proliferation and differentiation (Amariglio N, 2002).
Moreover, mu receptors are also expressed in human surgical specimens of both normal lung and small cell lung cancer cells (Arcuri et al 1999). Nitric oxide release is thought to be coupled with the mu3 receptor, thus suggesting endogenous morphine has a link to other biological processes, such as pain tolerance and addiction (Pryor et al 2005 Cadet 2004). This discovery has brought about the realisation that some of the discovered effects formerly attributed to nitrous oxide (NO) may in fact also be related to the effects of endogenous morphine. By exploring this morphine mediated signalling system that utilises nitric oxide, this may lead to new possibilities in the development of novel pharmaceuticals or even allow the redesign of old pharmaceuticals.
The role of endogenous morphine
Endogenous morphine production may be involved in autocrine, paracrine or hormonal regulation of immune, neural, nociceptive and cardiovascular pathways (Stefano et al 2000). Morphine is present in neurones and undergoes a calcium ion dependent release, which is consistent with it being a neurotransmitter or neuromodulator. A study, which examined the criteria for a substance to be classified as a neurotransmitter, in relation to endogenous morphine concluded that endogenous morphine did satisfy the criteria and it should be possible to class endogenous morphine as a neurotransmitter (Bianchi E, 2005). If morphine fits the criteria for a neurotransmitter then this goes to suggest that endogenous morphine has a biological function in humans.
Studies conducted by Harada et al have led to suggestion that endogenous morphine has various biological responses following surgery. This study demonstrated that endogenous morphine may have a role in stress response (Harada et al, 2000). This confirms that there may be a purpose for endogenous morphine in humans. Endogenous morphine may also be involved in the mechanism by which addictive substances, such as cigarettes, cause addiction (Casares et al 2006). This stimulatory effect identified in animals may also be present in humans, however, more studies are needed to prove this theory.
Other studies have linked morphine tolerance to the enhancement of glutaminergic neurotransmission, in particular, to increased function of the NMDA (N-methyl-D-aspartate) receptor. NMDA and glutamate are both neurotransmitters. Their link to morphine tolerance further suggests that morphine is endogenous in humans as morphine can interact with neurotransmitter receptors to exert a response (Joo et al 2006). Additionally, the neuropeptide orphanin FQ/nociceptin (OFQ/N) has been implicated in the sensation of pain, counteracting several effects of endogenous and exogenous opioids. This OFQ/N may act as an opioid-modulating agent involved in the development of morphine tolerance and dependence (Chung et al 2006).
Exercise stimulates the release of endogenous opioid peptides and increases pain thresholds in animal and human subjects (Rang et al 1998). Long term exercise brings about a reduction in sensitivity to morphine effects (Bailey et al 2006). A study by Yancey et al investigated whether exercise could increase endogenous opioid receptors. The study found that chronic exercise led to an increase in opioid tolerance due to the up-regulation of the mu opioid receptor (Yancey, 2004). This suggests that animals naturally produce endogenous opioids during exercise and these are similar to morphine as they seem to utilise the same opioid receptors and reinforce the possibility that morphine is an endogenous substance that can be synthesised by humans.
Normal human white blood cells have been shown to possess the ability to synthesise morphine. It has been shown that invertebrate neural tissue as well as human tissue, incubated with ethanol, cocaine or nicotine results in significant enhancement of morphine release (Sonetti et al 2005 & Kream et al 2006). This release of morphine demonstrates human tissue can secrete increased levels of morphine and suggests that the tissue can endogenously synthesise the morphine that is released.
Other physiological functions or endogenous morphine have also been suggested. Morphine has been shown to exert an immunosuppressive effect (Leon-Casasola 2004). Additionally, it seems morphine synthesis is increased following trauma and tissue damage (Cadet et al 2003). Functional endogenous opioid receptors have also been found in human sperm, indicating that endogenous morphine may have a role in the regulation of sperm physiology ( Agirregoitia E, 2006). These studies all strengthen the body of evidence which suggests endogenous morphine has many functions within humans.
CONCLUSION
There is increasing evidence to suggest that morphine can be synthesised endogenously in human tissue (Choi 2006). The discovery of opioid receptors, particularly the mu receptor, which binds morphine, demonstrates that the body can utilise opioid compounds for biological purposes (Scharrer et al 1994). It therefore seems logical that opioid compounds such as morphine have a purpose within the body. The discovery of agonists to endogenous opiate receptors does not conclusively show that these opioid substances are endogenously synthesised. More evidence is required in order to conclusively determine the exact biosynthetic pathway for morphine synthesis. The existence of an alternative L-DOPA pathway for morphine synthesis in humans has provided some evidence to support endogenous morphine production; however, more evidence is required.
There is a large body of evidence from both human and animal studies that shows how exercise can increase the number of opioid receptors in tissue, thus causing a morphine tolerance to develop (Bailey et al 2005). For this to occur then morphine must be involved in the natural response to chronic exercise. This may be one of the reasons why humans have endogenous morphine.
Morphine dependency studies have also further supported this theory as morphine seems to have an interaction with neurotransmitters within the body (Christie et al 2001). For this to occur then it would seem that morphine must have some biological function within the brain. Morphine also fits the criteria as a neurotransmitter itself, further suggesting that morphine is an endogenous substance.
For many years it was believed that morphine was introduced into the body through dietary sources. This can be disputed by the research carried out into the synthetic pathways for endogenous morphine. There are fundamental differences between the synthetic pathways identified in plants compared with the pathways investigated in humans. The utilisation of CYP2D6 isoenzyme in the synthesis of endogenous morphine seems to hold the key to explaining how animals and humans could synthesise their own endogenous morphine. There is also a step in the process which involves a substrate only present in animals. This therefore suggests that this synthetic pathway is unique to animals and the morphine produced by it could not have originated from a plant source.
Biosynthetic experiments on human neuroblastoma cells have shown that carcinoma cells have the ability to synthesise morphine (Poeaknapo et al 2005). If this is translated to normal human cells then this would provide further evidence for the ability of human cells to synthesise morphine. Furthermore, the precursors for endogenous morphine synthesis in human cell lines have also been identified as oxygen, tyramine, reticulin and thebane. It seems that humans’ cells have the required substrates to produce morphine.
In summary, the evidence shows that morphine is produced endogenously in humans. There have been several suggestions of its function or benefit to humans although knowledge in this area is not currently conclusive. Further studies will be required to conclusively show the physiological function of endogenous morphine in human beings.
