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First published online March 31, 2005
Journal of Experimental Biology 208, v-a (2005)
Copyright © 2005 The Company of Biologists Limited
doi: 10.1242/jeb.01592
Outside JEB |
NOVEL IN VITRO METHOD TO STUDY FISH INTESTINE
RSMAS, University of Miami
dmcdonald{at}rsmas.miami.edu
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Early in life, salmonid fishes move from a freshwater existence to a life in seawater, requiring a complete overhaul of their osmoregulatory systems. The vertebrate intestine is a major osmoregulatory organ and in fish, intestinal salt and water uptake is essential for maintaining water balance. Solute and water movement is mainly driven across the intestinal epithelium by the Na+, K+-ATPase, an ATP-requiring enzyme located on the basolateral membrane or blood side of the intestinal cell. Cortisol, a stress hormone, is thought to mediate the necessary osmoregulatory adaptations required for a salmonid's transformation between environments. It has been suggested that intestinal Na+, K+-ATPase activity, which becomes elevated in preparation for seawater survival, is stimulated by cortisol in many salmonid fishes. However, the influence of cortisol on fish intestine has never been measured directly. To determine the direct effects of cortisol on the intestine during salinity transfer, an in vitro intestinal tissue culture system is needed.
Thus, Philip Veillette and Graham Young set out to develop an intestinal tissue culture system from freshwater-adapted sockeye salmon Oncorhynchus nerka by adapting methods used for culturing salmon gill filaments. They decided to culture the pyloric ceca (appendages attached to the salmon's intestines) and the posterior intestine based on their significant roles in absorption and osmoregulation. They cut each intestinal region into pieces, incubated the intestinal preparations at 14°C and gassed them daily with a 95% O2, 5% CO2 mixture to keep the cells alive. To see if their tissue culture was still viable after 3 or 6 days in culture, Veillette and Young examined the preparations using light and transmission electron microscopy. Sure enough, they noticed intact nuclei and organelles, which meant that the intestinal cells were still functional; they had an in vitro intestinal tissue culture.
Veillette and Young could now test whether salmon intestinal Na+, K+-ATPase activity is stimulated by cortisol. In the absence of cortisol, the activity of Na+, K+-ATPase in the intestine preparation declined after several days in culture. However, when Veillette and Young treated the preparations with cortisol, they noticed significantly maintained Na+, K+-ATPase activity in both segments of the intestine, demonstrating for the first time that cortisol acts directly on the teleost intestine. The response to cortisol was dose-dependent and specific for cortisol over other corticosteroids within a physiological range. To see if they could replicate these results in vivo, they implanted salmon with slow-release cortisol, elevating plasma cortisol concentrations in these fish. Just as they had found in vitro, cortisol increased Na+, K+ ATPase activity in both intestinal segments in live fish.
Using a novel approach, Veillette and Young have shown that the salmon pyloric ceca and the posterior segment of the intestine are direct targets for the osmoregulatory actions of cortisol in salmonids. Their novel culture system could potentially be used to examine the effects of a variety of neuroendocrine factors on the intestine under many different physiological conditions.
References
Veillette, P. A. and Young, G. (2005). Tissue
culture of sockeye salmon intestine: functional response of Na+,
K+-ATPase to cortisol. Am. J. Physiol. Regul. Integr.
Comp. Physiol. (February 3, 2005). Articles in Press doi:10.1152/ajpregu.00741.2004
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