No abstract

We have analyzed the effects of different native membrane lipid composition on the thermodynamic properties of the Na+-K+-ATPase in different epithelia from the gilthead seabream Sparus aurata. Thermodynamic parameters of activation for the Na+-K+-ATPase, as well as contents of lipid classes and fatty acids from polar lipids were determined for gill epithelia and enterocytes isolated from pyloric caeca, anterior intestine and posterior intestine. Arrhenius analyses of control animals revealed differences in thermal discontinuity values (Td) and activation energies determined at both sides of Td between intestinal and gill epithelia. Eyring plots disclosed important differences in enthalpy of activation (ΔH‡) and entropy of activation (ΔS‡) between enterocytes and branchial cells. Induction of n-3 LCPUFA deficiency dramatically altered membrane lipid composition in enterocytes, being the most dramatic changes the increase in 18:1n-9 (oleic acid) and the reduction of n-3 LCPUFA (mainly DHA, docosahexaenoic acid). Strikingly, branchial cells were much more resistant to diet-induced lipid alterations than enterocytes, indicating the existence of potent lipostatic mechanisms preserving membrane lipid matrix in gill epithelia. Paralleling lipid alterations, values of Ea1, ΔH‡ and ΔS‡ for the Na+-K+-ATPase were all increased, while Td values vanished, in LCPUFA deficient enterocytes. In turn, Differences in thermodynamic parameters were highly correlated with specific changes in fatty acids, but not with individual lipid classes including cholesterol in vivo. Thus, Td was positively related to 18:1n-9 and negatively to DHA. Td, Ea1 and ΔH‡ were exponentially related to DHA/18:1n-9 ratio. The exponential nature of these relationships highlights the strong impact of subtle changes in the contents of oleic acid and DHA in setting the thermodynamic properties of epithelial Na+-K+-ATPase in vivo. The effects are consistent with physical effects on the lipid membrane surrounding the enzyme as well as with direct interactions with the Na+-K+-ATPase.

SUMMARYThe regulation of neutral amino acid transport by cyclic AMP (cAMP) and calcium across the isolated duodenum of the lizard Gallotia gallotihas been studied under short-circuit conditions. Active L-alanine transport was stimulated by forskolin, theophylline and dibutyryl cyclic AMP (db-cAMP). All these agents increased transmural potential difference (PD) and short-circuit current (Isc) in a manner consistent with the activation of a chloride secretory pathway. Both forskolin and theophylline increased intracellular cAMP levels in the lizard duodenal mucosa. Addition of calcium ionophore A23187 rapidly reduced mucosa-to-serosa L-alanine fluxes and diminished net L-alanine transport. Despite the reduction of alanine fluxes by A23187, transepithelial PD and Iscvalues were increased by the ionophore. Analyses of the responses of isolated transport pathways indicated that the Na+-independent L-alanine transport system was unaffected by db-cAMP or calcium ionophore. By contrast,Na+-dependent transport activities were profoundly modified by these agents. Thus, while system A [α-methylamino-isobutiric acid(MeAIB)-transporting pathway] was stimulated by increased calcium, system ASC activity was nearly abolished. Calcium ionophore also potentiated the electrogenic response of system A. Forskolin strongly stimulated system ASC activity but left system A activity unchanged. Activation of system ASC by forskolin was clearly electroneutral, as pre-incubation of the tissues with the chloride channel blocker diphenylamine-2-carboxilic acid (DPC) completely prevented forskolin-induced transepithelial electrical responses. It is concluded that intracellular messengers cAMP and calcium oppositely modulate active Na+-dependent L-alanine transport in the lizard intestine. The different sensitivity exhibited by individual transport pathways may well account for the changes observed in overall alanine transport.