It is well established that H<sup>+</sup>-coupled dipeptide transport in the cultured intestinal cell line, Caco-2 is electrogenic and results in intracellular acidification, via the apically located transport protein, PepTl. The purpose of this study was to initially investigate both the pH dependency and electrogenic nature of H<sup>+</sup>-coupled dipeptide transport, using microspectrofluorometric analysis of intracellular pH and short circuit current (I<sub>SC</sub>) measurement of Gly-Sar transport in Caco-2 monolayers. This aimed to characterise both the apical and basolateral transport mechanisms involved in H<sup>+</sup>-coupled Gly-Sar transport under normal physiological conditions and subsequently investigate how these mechanisms are affected by exposure to pathophysiological manipulators. Transepithelial H<sup>+</sup>-coupled Gly-Sar transport was saturable and displayed typical Michaelis-Menton kinetics in Na<sup>+</sup>-free Krebs buffer. In experiments using an isotonic mannitol buffer, apical Gly-Sar induced similar pH dependent transport kinetics. While the characteristics of basolateral Gly-Sar transport were distinct from PepT1 they were also pH dependent and saturable, indicating that protons are responsible for charge carriage during transepithelial dipeptide transport, via the apical transporter, PepT1 and a kinetically distinct pH-dependent basolateral transporter. The heat stable enterotoxin of <i>E. coli</i>, STa inhibited the capacity of Gly-Sar transport in Caco-2 epithelia as well as inhibiting the Na<sup>+</sup>-dependent recovery from Gly-Sar induced acidification. These effects were also observed with the membrane permeable analogue of cGMP, 8-Br-cGMP, indicating that STa-induced inhibition of H<sup>+</sup>-coupled Gly-Sar transport was mediated via elevation of intracellular cGMP levels. The disruption of STa-dependent, but not 8-Br-cGMP-dependent inhibition of Gly-Sar induced I<sub>SC</sub> by 21CIAdo, confirmed this proposition. Additionally, the NO donor, SNP, inhibition Gly-Sar-induced I<sub>SC</sub>, with characteristics similar to those observed with 8-Br-cGMP. Using protein kinase inhibitors it emerged that 8-Br-cGMP-dependent inhibition of Gly-Sar transport involved both PKG and PKC, while only PKG was implicated in the STa-dependent inhibition of Gly-Sar transport in these cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:248618 |
| Date | January 2002 |
| Creators | Marsh, Susan E. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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