greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This talk will focus on the recent molecular advances for Na+ uptake in freshwater fish and the relative roles of NHEs and Na+ channels in Na+ transport and acid–base regulation in freshwater fishes. Emphasis will be placed on thermo- dynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermody- namic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner. doi:10.1016/j.cbpa.2008.04.215 A5.26 What is the active corticosteroid in primitive aganthan vertebrates? The effects of corticosteroids on gluconeogenesis and magnesium regulation in the Pacific hagfish (Eptatretus stouti) N. Bury (King's College London); J. Ede (University of Alberta); G. Goss (University of Alberta) The corticosteroid receptors (CR) control a vast array of physiolo- gical processes acting primarily as ligand-dependent transcription factors. The origins of the gnathostomata CRs can be traced back to an ancestral steroid receptor present in a primitive agnathan vertebrate, and gene or genome duplications events have given rise to a glucocorticoid (GR) and mineralocortioicd receptors (MR) present in mammals and fish. Recent studies have shown that the CR in the agnathans show transactivational characteristics of the tetrapod MR. However, there has been little work conducted on the physiological role of corticosteroids in the agnathan group the myxini (hagfish). In the present study Pacific hagfish (Eptatretus stouti) were injected in their intraperitoneal cavity a bolus of coconut oil containing 0, 20, 100 or 200 mg/kg of either cortisol, corticosterone or 11-deoxycorticosterone (DOC). Blood samples were taken on days 4 and 7 for plasma cortisol, and glucose measurements. In an additional experiment hagfish were injected with 100 mg/kg of either cortisol or 11-deoxycorticosterone and on the 5th day following injection the hagfish were transferred to seawater containing 50 mM MgCl 2 to assess their ability to regulate Mg. Results show that fish treated with DOC showed a greater ability to deal with the MgCl 2 challenge, and also showed evidence of increase plasma glucose concentrations on Day 7. These results suggest that DOC, and not cortisol, is the active corticosteroid potentially having gluconeogenesis and mineralocorticoid activity in hagfish. However, other physiological endpoints are needed to verify the physiological role of this hormone and receptor. doi:10.1016/j.cbpa.2008.04.216 A5.27 Calcium regulation in the European flounder, Platichthys flesus M. Greenwood, R. Balment (The University of Manchester) The potential role of the calcium sensing receptor (CaSR) in the regulation of stanniocalcin (STC) secretion from the corpuscles of Stannius (CS) has been investigated. The secretion of parathyroid hormone from mammalian parathyroid glands is tightly regulated by freely ionised calcium, through interactions with the CaSR. To investigate tissue sensitivity to calcium in fish I have cloned and identified sites of mRNA and protein expression of the flounder CaSR. Interestingly, CaSR mRNA expression was highest in the CS with descending levels in the caudal neurosecretory system (CNSS) b bladder b gill b kidney b skin b brain and intestine. The high level of CaSR expression in the CS corresponded with high levels of STC mRNA expression, suggesting a regulatory role in STC secretion. Furthermore, high levels of CaSR were also found in the Dahlgren cells of the CNSS where STC was also highly expressed. Immunofluorescent protein localisation studies showed the CaSR to be highly expressed in STC positive CS cells, gill chloride cells and at the apical membranes of renal proximal tubules. There were no differences in CS CaSR mRNA expression when comparing fish chronically (14 days) adapted to seawater, freshwater or nominally deionised water. However, STC mRNA expression showed sensitivity to environmental salinity with significantly lower levels in freshwater and nominally deionised water by comparison with seawater fish. These findings suggest similar sensitivity of CS cells to calcium in seawater, freshwater and nominally deionised water fish. Interestingly, CNSS STC mRNA expression also showed sensitivity to environmental salinity. This adds to emerging evidence from our laboratory suggesting that changes in CNSS STC expression potentially impact on calcium regulation. doi:10.1016/j.cbpa.2008.04.217 A5.28 Building a house when supplies are minimal: Ca 2+ and HCO 3 - acquisition in freshwater by embryos of the common pond snail Lymnaea stagnalis S. Ebanks, M. Grosell (University of Miami, Rosenstiel School of Marine and Atmospheric Science) The inherent challenge faced by freshwater organisms to maintain hyperosmotic internal conditions against a steep ionic gradient by active ion transport has been studied extensively in many species. Adult common pond snails, Lymnaea stagnalis, appear similar to most studied freshwater organisms with respect to Na + and Cl - balance. However, the developing embryos of this species exhibit ion-specific dependence on environmental availability for shell-forming ions, specifically Ca 2+ and HCO 3 - . Our objectives are to evaluate the sources and mechanisms for acquiring these shell-forming ions considering the relative ionic poverty of freshwater systems. These objectives are pursued by measurements of Ca 2+ and titratable acid fluxes and by assessment of the importance of water chemistry to shell-formation during post-metamorphic embryonic development. These objectives are addressed by utilizing developmental observations and physiolo- gical endpoints across a range of water [Ca 2+ ] [HCO 3 - ] (Ca 2+ - and HCO 3 - - free concentrations ≤ 1% of controls). Results thus far indicate that embryonic development is retarded in waters without added Ca 2+ and HCO 3 - . Time to hatch under normal conditions is 8–10 days; however, in nominally “Ca 2+ -free” media, embryos failed to hatch, even after 15 days. Requirements for Ca 2+ and HCO 3 - are apparently coordinated with metamorphosis at approximately 5 days post-oviposi- tion. In contrast to the demonstrated Ca 2+ requirements, multiple independent experiments corroborate that these post-metamorphic embryos (onset of shell formation evident) are primarily obtaining CO 3 2- for shell formation from metabolic CO 2 production, which hinges on S104 Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S97–S114