The active Na(+)-independent transport of L-alanine across the duodenal mucosa of the lizard Gallotia galloti was studied in Ussing-type chambers using a computer-controlled voltage clamp. Addition of L-alanine to the Na(+)-free bathing solutions resulted in a significant L-alanine absorption (J(net)) that was paralleled by an increase in transepithelial short-circuit current (I(sc)) and potential difference (PD) without apparent changes in the tissue conductance. The concentration dependence of J(net), PD, and I(sc) displayed Michaelis-Menten kinetics. L-alanine-induced electrical changes were completely inhibited by external alkaline pH or by the H(+)-ionophore carbonyl cyanide m-chlorophenyl-hydrazone in the bathing solution. The alanine-induced electrogenicity was dependent on the presence of extracellular K(+) and could be blocked by serosal Ba(2+) or mucosal orthovanadate. These results suggest the existence of an H(+)-coupled L-alanine cotransport at the apical membrane of enterocytes. The favorable H(+) driving force is likely to be maintained by an apical vanadate-sensitive H(+)-K(+)-ATPase, allowing the extrusion of H(+) in an exchange with K(+). Potassium exit through a basolateral barium-sensitive conductance provides the key step for the electrogenicity of L-alanine absorption.

L-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net L-alanine fluxes, transepithelial potential difference (PD), and short-circuit current (Isc) showed concentration-dependent relationships. Na(+)-dependent L-alanine transport was substantially inhibited by the analog alpha-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited by L-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K(m) values. Na(+)-dependent L-alanine transport, but not MeAIB transport, was partially inhibited by L-serine and L-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for L-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the L-alanine-induced electrogenicity. It is concluded from the present study that the active Na(+)-dependent L-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.

Rumen-protected conjugated linoleic acid (CLA) reportedly improves fertility in lactating dairy cows by reducing the postpartum interval to first ovulation and enhancing the circulating insulin-like growth factor-I (IGF-I) levels. The objectives of this study were to evaluate the blood metabolites, hormones, follicular fluid (FF) and liver for the effect of CLA supplementation (50 g top-dressed daily from 15 days pre-partum to 65 days in milk -DIM -). Pre-partum Holstein cows (n = 24) were assigned to two treatments: a Control and CLA group (n = 12 cows/group). Dry matter intake (DMI) and milk production were recorded daily. At 26 DIM, ovulation was synchronized and at 34 DIM, plasma and FF were analysed for paraoxonase 1(PON) levels. Moreover plasma was analysed for IGF binding protein 2 and 3 (IGFBP). From 34 DIM, blood samples and FF from follicles >9 mm were collected and analysed for estradiol, progesterone, IGF-I and lipoproteins. A liver biopsy was performed at 65 DIM and analysed for the expression of IGF-I, growth hormone receptor (GHR), pyruvate carboxylase (PC) and cytosolic phosphoenolpyruvate carboxykinase (PECK). CLA supplemented cows, compared to the control group, recorded a significant lower milk fat production, improved DMI and energy balance and recorded significant increased plasma concentrations of IGF-I, cholesterol, low density lipoprotein (LDL) and IGFBP-3 (interaction treatment x DIM). The concentration of IGF-I, high density lipoprotein (HDL) and LDL tended to be higher in FF than plasma. This study confirms the positive effects of dietary CLA supplementation on the metabolism, by improving the energy intake and reducing the negative energy balance. Moreover, the improvement of plasma IGF-I levels observed in this study, coupled with a better energy balance support previous studies showing a positive effect of CLA supplementation on reproduction. However, CLA did not alter the plasma and the FF concentration of PON, nor the liver gene expression. _______________________________________________________________________________________

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.