Inhibition of intestinal dipeptide transport by the neuropeptide VIP is an anti‐absorptive effect via the VPAC₁ receptor in a human enterocyte‐like cell line (Caco‐2)

Abstract

1. Optimal dipeptide and peptidomimetic drug transport across the intestinal mucosal surface is dependent upon the co-operative functional activity of the di/tripeptide transporter hPepT1 and the Na(+)/H(+) exchanger NHE3. The ability of the anti-absorptive enteric neuropeptide VIP (vasoactive intestinal peptide) to modulate dipeptide uptake was determined using human intestinal (Caco-2) epithelial cell monolayers. 2. Uptake of glycylsarcosine (Gly-Sar) across the apical membrane of Caco-2 cell monolayers is inhibited by basolateral exposure to either VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), or the VPAC(1) receptor agonist [(11,22,28)Ala]-VIP. Inhibition of Gly-Sar uptake is observed only in the presence of extracellular Na(+). Reverse-transcription polymerase chain reaction (RT-PCR) demonstrates that VPAC(1) mRNA is expressed in Caco-2 cells whereas VPAC(2) mRNA is not detected. 3. The VIP-induced inhibition of Gly-Sar uptake is abolished in the presence of the protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl). 4. (22)Na(+) uptake across the apical membrane is inhibited by the selective NHE3 inhibitor S1611. Experiments with BCECF [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that VIP reduces the NHE3-dependent recovery of intracellular pH (pH(i)) after dipeptide-induced acidification. Western blot of Caco-2 cell protein demonstrates expression of the NHE regulatory factor NHERF1 (expression of which is thought to be required for PKA-mediated inhibition of NHE3). 5. VIP has no effect on Gly-Sar uptake in the presence of S1611 suggesting that VIP and S1611 both modulate dipeptide uptake via the same mechanism. 6. These observations demonstrate that VIP (and PACAP) modulate activity of the H(+)/dipeptide transporter hPepT1 in a Na(+)-dependent manner consistent with the modulation being indirect through inhibition of NHE3.

Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells

“…It has been reported that PEPT1, a proton-dependent dipeptide transporter, is linked to Na/H exchange in mammalian intestinal epithelial cells to regulate cellular pH (1,2,11,12,28) as well as the activation of other proton-solute cotransporter processes to stimulate Na/H exchange in intestinal epithelial cells (27). Interestingly, colon cancer cells (Caco-2) transfected with SGLT1 immediately upon activation have been suggested to stimulate Na/H exchange.…”

Regulation of drug transporters by PDZ adaptor proteins and nuclear receptors

“…Indeed, uptake of dipeptides by the H + /oligopeptide transporter on the apical membrane in Caco-2 cells has been suggested to be indirectly controlled by NHEs, which supply the H + gradient for this peptide uptake mechanism(s) (Thwaites et al, 2002). Hence, this oligopeptide transport system exhibits apparent Na + -dependence in its substrate uptake, and NHE inhibitors hinder uptake of glycylsarcosine (GlySar) in Caco-2 cells (Thwaites et al, 2002;Anderson et al, 2003). Functional modulation by NHE can also occur intracellularly, since NHE can export H + , which is taken up by oligopeptide transporters.…”

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

“…Cell culture of Caco-2 cells (passage 105-124) was performed essentially as described previously [12,20,21]. Experiments were performed 14-17 days after seeding and 18-24 h after feeding.…”

“…However, there are differences in the ionic dependence of peptide transport when hPepT1 activity is compared in isolation and in the endogenous situation where hPepT1 is colocalised in the specialised brush-border membrane with a large number and variety of transport proteins involved in transmembrane solute and ion movement. Following heterologous expression in X. laevis oocytes or HeLa cells, PepT1 functions as an H + -coupled, pHdependent, Na + -independent transporter [13,14,16], whereas in intact intestinal tissue preparations dipeptide uptake is also dependent on extracellular Na + [16][17][18][19][20][21]. The apparent Na + dependence of dipeptide uptake is the net result of a reciprocal functional relationship between PepT1 and an apically localised Na + /H + exchanger [20,22,23].…”

“…NHE3 is known to be down-regulated by activation of the protein kinase A (PKA) pathway. We have been able to mimic this response pharmacologically and have demonstrated that incubation of Caco-2 cell monolayers with forskolin, 8-Br-cAMP or activators of the basolateral VPAC 1 receptor leads to an inhibition in NHE3 activity and a concomitant reduction in apical Gly-Sar uptake via hPepT1 [20,21]. Activation of the PKA pathway has no effect on Gly-Sar uptake under conditions in which NHE3 is inactive, e.g.…”