OBJECTIVES: The study was designed to determine if morphine was present in lobster tissues. It was also important to determine, as in other animals, if its levels would change in response to stress. In this regard, it was also important to determine if lobster immune and neural tissues express the mu opiate receptor subtype, which was coupled to constitutive nitric oxide synthase derived nitric oxide release.
METHODS: Homarus americanus were used in these experiments. Morphine was purified in lobster tissues via high pressure liquid chromatography coupled to UV detection. It was quantified via radioimmunoassay (RIA) and was identified via quadruple time of flight - mass spectrometry. Animals were subject to 2 forms of trauma, namely pereiopod-ablation or lipopolysaccaride (LPS) - injection, and morphine levels determined in nerve cord or hemolymph. Real-time nitric oxide production was determined via an amperometric probe. RT-PCR was used to determine the presence of a micro opiate receptor transcript.
RESULTS: In Homarus americanus hemolymph and nerve cord morphine was found. RIA revealed morphine levels of 3.36 pg/mg +/ - 0.48 SEM (N=8) in nerve cord and 717.88 pg/ml +/ - 56.77 SEM (N=58) in hemolymph. In stressed (pereiopod-ablated or LPS-injected) animals, the endogenous morphine levels initially increased significantly by 24% for hemolymph and 48% for nerve cord. By day 5, the stressed and control values for endogenous morphine, in both tissues, was lower and non-distinguishable. In both hemocytes and neural cells, morphine, not met-enkephalin, stimulated constitutive nitric oxide release in a naloxone antagonizable manner, demonstrating a mu opiate receptor mediated phenomenon and suggesting the presence of the mu opiate receptor subtype, micro3, since it is opiate alkaloid selective and opioid peptide insensitive. RT-PCR revealed the presence of a micro opiate receptor transcript in Homarus neural and immune tissues, which exhibits a 100% sequence identity with its human counterpart.
CONCLUSION: Taken together, after eliminating all sources of contamination, morphine is present in lobster tissues, potentially demonstrating hormonal and neurotransmitter functions that are involved in the animals' stress response.