The Role of Polyamines in Osmotic Stress Tolerance in Gulf Killifish Fundulus Grandis

Abstract

Polyamines are a diverse class of aliphatic molecules that are highly polycationic at physiological intracellular pH. Polyamine levels are regulated by the coordinated actions of arginase (Arg), ornithine decarboxylase (Odc), and polyamine oxidase (Pao). Although little is known of their functions in fish, polyamines have been implicated in diverse physiological processes, including RNA transcription, cell growth, cell proliferation, and cell apoptosis. The main objective of this study was to describe the transcription and enzymatic activities of key enzymes for polyamine metabolism, to measure polyamine levels, and to assess putative roles of polyamines in the gills of the Gulf killifish (Fundulus grandis) during hypoosmotic challenge. In addition, the influence of irreversible inhibition of Odc by alpha-DL-difluoromethylornithine (DFMO) was assessed in the gills. Furthermore, the transcription and enzymatic activities of Arg, Odc and Pao was assessed in other tissues such as intestine and liver during hypoosmoitc challenges. Adult F. grandis were reared in 5 ppt and acutely transferred to 5, 2, 1, 0.5, and 0.1 ppt water, and gills were sampled at 6 h, 1 d, 3 d, and 7 d post-transfer. Results showed that arg II and odc mRNA levels were highly upregulated in the gills during the first few days post-transfer to hypoosmotic water. Hypoosmotic exposure also produced increases in gill Odc activity and in the concentrations of putrescine, spermidine, and spermine. DFMO application inhibited Odc activity and reduced polyamine levels after hypoosmotic exposure (0.1 ppt). Although gill putrescine levels remained elevated throughout the first 7 d post transfer to 0.1 ppt, the concentrations of spermidine and spermine decreased over time. The ratio of putrescine level over the sum levels of spermidine and spermine increased after 0.1 ppt exposure at 1 d and beyond. Concomitant with freshwater acclimation, an increase in Pao activity suggested that polyamine catabolism was upregulated in the gills. The phenotype of mitochondrion-rich cells (MRCs) in the gill epithelium shifted from a seawater type to a freshwater type following transfer to 0.1 ppt water in correlation with the increase in mRNA levels of arg II and odc in MRCs. In addition, the isolated opercular epithelium pretreated with spermidine had a lower active Cl- secretion rate and membrane conductance following symmetrical hypotonic exposure. Transcription and enzymatic activities of Arg II, Odc, and Pao were upregulated in the intestine and liver during hypoosmotic exposure, suggesting that polyamine levels are regulated in multiple tissues of the killifish. The putative roles