Comparative Biochemistry and Physiology Part B 125 (2000) 483 – 491 Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida Clare E. Morrall a,b, *, Tamara S. Galloway a , Henry G. Trapido-Rosenthal b , Michael H. Depledge a a Plymouth Enironmental Research Centre, Uniersity of Plymouth, Plymouth, PL48AA, UK b Bermuda Biological Station for Research, Ferry Reach, St. George’s, GE O1, Bermuda Received 27 July 1999; received in revised form 16 November 1999; accepted 20 December 1999 Abstract The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [ 3 H]arginine to [ 3 H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N G -methyl-L-arginine (L-NMA), but not the arginase inhibitors L-valine and L-ornithine. NOS activity was predomi- nantly cytosolic, and was characterised by a K m for arginine of 19.05 M and a V max of 2.96 pmol/min per g protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Citrulline assay; Cnidaria; Coral; Invertebrate; NADPH diaphorase; Nitric oxide synthase; Nitric oxide; Sea anemone www.elsevier.com/locate/cbpb 1. Introduction Nitric oxide (NO) is a multi-functional messen- ger that has been identified in the majority of animal phyla, in bacteria and in a variety of plant species. NO has been implicated in many funda- mental cellular processes (Nathan and Xie, 1994) and is generated by a family of nitric oxide syn- thase (NOS) isozymes which differ in their mode of expression, primary sequence and calcium de- pendency (Moncada and Higgs, 1993). These dif- ferent isoforms have been characterised in mammalian species. A constitutive Ca 2 + /calmod- ulin-dependent cytosolic isoform, nNOS, has been located in neuronal tissues, and subsequently also in skeletal muscle, epithelial cells and other tissues (Griffiths and Stuehr, 1995). The Ca 2 + -dependent membrane-associated form, eNOS, is found in vascular endothelial cells and is activated by Ca 2 + influx, agonists such as bradykinin and by shear stress (Jansenns et al., 1992). A third Ca 2 + - independent isoform is induced in response to bacterial lipopolysaccharide (LPS) and a variety of cytokines, notably interleukin-1 and interferon-  (Stuehr, 1997). It is found primarily in macrophages, but is also induced in other tissues e.g. endothelial cells and hepatocytes. Constitutive expression of iNOS may also occur in tissues such as lung epithelium (Nathan and Xie, 1994). Whilst calmodulin is permanently bound to iNOS, it is * Corresponding author. Tel.: +44-1-2971880; fax: +441- 2978143. E-mail address: cmorrall@bbsr.edu (C.E. Morrall) 0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII:S0305-0491(00)00157-7