Dopamine in the ink defence system of Sepia officinalis: biosynthesis, vesicular compartmentation in mature ink gland cells, nitric oxide (NO)/cGMP-induced depletion and fate in secreted ink.

The biosynthesis, localization, and fate of catecholamines in the ink gland of the cuttlefish S. officinalis were investigated by combined biochem. and immunohistocytochem. methodologies. HPLC anal. of crude ink gland exts. indicated the presence of dopa (2.18 nmol/mg protein) and DA (dopamine, 0.06 nmol/mg protein), but no detectable noradrenaline or adrenaline. DA was shown to derive from L-tyrosine, according to expts. 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 exts., and the presence of the enzyme was confirmed by Western-blot anal. 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) signaling pathway, as apparent from incubation expts. 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. Immunohistochem. results coupled with electron microscopy indicated that DA was concd. in vesicles specifically localized in the mature melanin-producing cells of the ink gland proximal to the lumen and sepd. from the melanin-contg. 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 assocd. with the melanin granules, due apparently to phys. adsorption. Overall, these results point to DA as a marker of cell maturation in Sepia ink gland subject to release by the NO/cGMP signaling pathway, and disclose apparently overlooked DA-melanin interactions in secreted ink of possible relevance to the defense mechanism.