The biosynthesis, localization and fate of catecholamines in the ink gland of the cuttlefish Sepia officinalis were investigated by combined biochemical and immunohistocytochemical methodologies. HPLC analysis of crude ink gland extracts indicated the presence of dopa (2.18±0.82 nmol/mg of protein) and DA (dopamine, 0.06±0.02 nmol/mg of protein), but no detectable noradrenaline or adrenaline. DA was shown to derive from l-tyrosine, according to experiments performed by incubating intact ink glands with [l-14C]tyrosine. The biosynthetic process involves a tyrosine hydroxylase and a dopa decarboxylase pathway and is independent of tyrosinase. The tyrosine hydroxylase activity was detected under conditions of tyrosinase suppression in the cytosolic fraction, but not in the melanosomal fraction, of ink gland extracts, and the presence of the enzyme was confirmed by Western-blot analysis. Dopa and DA were found to be released from the ink glands by processes controlled through the NMDA-nitric oxide-cGMP (where NMDA stands for N-methyl-d-aspartate) signalling pathway, as apparent from incubation experiments performed with [l-14C]tyrosine in the presence of NMDA, diethylamine NONOate (diethylamine diazeniumdiolate), a nitric oxide donor, 8-bromo-cGMP or a guanylyl cyclase inhibitor. Immunohistochemical results coupled with electron microscopy indicated that DA was concentrated in vesicles specifically localized in the mature melanin-producing cells of the ink gland proximal to the lumen and separated from the melanin-containing melanosomes. NMDA receptor stimulation or exposure to an NO donor caused a marked loss of DA immunoreactivity in mature cells, consistent with a release process. In the lumen of the ink gland, where mature exhausted cells pour their contents, DA immunoreactivity was found to be associated with the melanin granules, due apparently to physical adsorption. Overall, these results point to DA as a marker of cell maturation in Sepia ink gland subject to release by the NO/cGMP signalling pathway, and disclose apparently overlooked DA–melanin interactions in secreted ink of possible relevance to the defence mechanism.
Abbreviations used: BH4, (6R)-5,6,7,8-tetrahydro-l-biopterin dihydrochloride; 8-Br-cGMP, 8-bromo-cGMP; DA, dopamine; DEA/NO, 2-(N,N-diethylamino)-diazenolate-2-oxide; DTT, dithiothreitol; NMDA, N-methyl-d-aspartate; ODQ, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one.
Dedicated to the memory of Professor Giuseppe Prota (1938–2003).
Present address: Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.