Membrane mechanisms for electrogenic Na⁺-independentl-alanine transport in the lizard duodenal mucosa

Abstract

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.

Electrogenic Na+-dependentl-alanine transport in the lizard duodenum. Involvement of systems A and ASC

“…For neutral amino acids, the existence of systems that transport [NBB system (neutral brush border system, transports most neutral amino acids but excludes MeAIB); system A; system ASC; Phe system (primarily phenylalanine, glycine, and methionine); ␤-amino (␤-amino acids such as taurine and ␤-alanine); Imino (imino acids such as proline, hydroxyproline); and the Na ϩ -independent system L] have been reported in isolated cells or membrane vesicles obtained from the small intestine of different species of mammals (11,19,30). Recently, we have demonstrated the presence both of Na ϩ -dependent and -independent active-transport mechanisms for L-alanine in the duodenum of the lizard Gallotia galloti (13).…”

Regulation of L-alanine transport systems A and ASC by cyclic AMP and calcium in a reptilian duodenal model

“…This conclusion is supported by our data of unchanged unilateral fluxes from the serosal compartment. Using electrophysiological and radiotracer techniques, we have recently demonstrated that active L-alanine transport across the lizard duodenum could be explained by the simultaneous operation of different carriers (Díaz et al, 2000;Medina et al, 2001). These distinct transport pathways could be resolved on the basis of their different Na + dependence, substrate affinity, kinetic features and electrical properties.…”

“…We have previously demonstrated that, under short-circuit conditions, the overall L-alanine transport in the lizard duodenum could be completely explained by the simultaneous participation of two Na + -dependent transport systems, endowed with properties of systems ASC and A, plus one Na + -independent electrogenic carrier (Díaz et al, 2000;Medina et al, 2001). These three different transport systems could be individually dissected by means of a strategy based on the differential substrate affinities and sodium dependence.…”

“…First, we measured L-alanine fluxes under short-circuit conditions using Na + -free solutions. Under these conditions, active L-alanine transport was carried by an electrogenic cycloleucineand 2-aminobicyclo-(2,2,1)-heptane-2-carboxilic acid (BCH)-sensitive pathway (Díaz et al, 2000). The results shown in Figs·4A and 5A indicate that neither dbcAMP nor A23187 affected unidirectional or net L-alanine fluxes, which strongly suggests that changes in the activity of the Na + -independent carrier were not responsible for the effects on the overall L-alanine transport observed above, but rather these resulted from the activities of the Na + -dependent systems.…”

“…This epithelium has been studied over the past two decades, and the different pathways for neutral amino acid transport have been recently identified and characterised physiologically (Díaz et al, 2000;Medina et al, 2001). …”