It is well documented that glutamine is an important nutrient required for the normal growth and differentiation of intestinal enterocytes [
23,
24]. Therefore, glutamine becomes more important to help restore the mucosal integrity and function of the small intestinal mucosa in chronic pathophysiological conditions [
23]. There are two different Na dependent co-transport pathways that enable glutamine absorption in the rabbit intestine, namely B0AT1 in villus and SN2/SNAT5 in crypt cells [
7,
8]. It has been shown previously that B0AT1 is inhibited in villus during chronic intestinal inflammation, whereas SN2/SNAT5 is stimulated in crypt cells [
9] and treatment with a broad spectrum immune modulator reversed these alterations during chronic intestinal inflammation [
19]. In the chronically inflamed intestine, mast cells have been shown to be important in the pathogenesis and progression of disease [
14,
25]. However, the regulation of Na-glutamine co-transporter alterations by mucosal mast cells during intestinal inflammation is not known. Therefore, the aim of this study was to investigate the role of the mucosal mast cells on Na-glutamine co-transport changes during chronic intestinal inflammation.
In the present study, mast cell stabilization by ketotifen reversed the inhibition of B0AT1 in intestinal villus during chronic intestinal inflammation and the mechanism of reversal of inhibition was secondary to restoration of diminished co-transporter numbers in the villus cell BBM. The mechanism of B0AT1 inhibition in the chronically inflamed intestine, secondary to a reduction in co-transporter numbers in the villus cell BBM, appears to be unique to this co-transporter. This is because other villus cell BBM Na-amino acid and di-peptide transporters are affected by a different mechanism during chronic intestinal inflammation. We have previously shown that Na-alanine co-transport (mediated by ATB0) in villus BBM of the rabbit small intestine is inhibited during chronic intestinal inflammation, not secondary to alteration in the number of BBM co-transporters, but secondary to a reduction in the affinity of the co-transporter for alanine [
26]. Similarly, the proton dipeptide co-transporter (PEPT1) was also significantly diminished during chronic intestinal inflammation and the mechanism of this inhibition was again secondary to a reduction in the affinity of the co-transporter rather than an alteration in the number of co-transporters on the BBM of villus cells [
18]. However, a different class of Na-nutrient co-transporters, specifically, Na-glucose co-transporter SGLT1, is inhibited during chronic intestinal inflammation by a reduction in the number of BBM co-transporters [
27].
Unlike villus cells, ketotifen reversed the stimulation of SN2/SNAT5 in crypt cells and the mechanism of reversal was secondary to the restoration of the affinity (
K
m
) of the co-transporter for glutamine. SN2/SNAT5 is the only Na-nutrient co-transporter that has been reported to be present in the BBM of crypt cells from the rabbit small intestine [
8]. Numerous studies demonstrate that Na-glucose [
18] Na-alanine [
17], Na-bile acid co-transport [
16] and PEPT1 [
25] are present only in villus cells, but not crypt cells in the rabbit intestine [
16-
18]. Thus, presence of Na-glutamine co-transport in crypt cells further emphasizes the importance of this nutrient for the well-being of intestinal epithelial cells. Further, unlike all other Na-nutrient co-transport processes which are inhibited during chronic intestinal inflammation [
16,
18,
28], only SN2/SNAT5 is stimulated so as to potentially compensate for the inhibition of Na-glutamine co-transport in villus cells [
9]. Though the reversal of Na-K-ATPase activity in ketotifen treated inflamed villus and crypt might appear to be partially responsible for the reversal of B0AT1 and SN2 activities, BBMV vesicle kinetic studies and Western Blot analyses suggest that they are directly regulated by specific immune inflammatory mediators. The differential mechanism of regulation of Na-glutamine co-transport in crypt as compared to villus, specifically, altered affinity of SN2/SNAT5 during chronic intestinal inflammation suggests that a specific immune inflammatory mediator/pathway might be responsible for each Na-glutamine co-transport alteration [
29]. Activated mast cells are known to generate and release abundant quantities of preformed mediators such as tryptase, histamine, serotonins, proteases, proteoglycans and cytokines such as tumor necrosis factor α (TNFα) [
30,
31]. TNFα has been shown to trigger multiple signaling pathways that regulate innumerable target proteins. More specifically, it has been shown to regulate several transcription factor proteins such as nuclear factor kappa B and activating protein 1 [
32]. In the present study, alteration of the transcription factors by TNFα activated signaling pathways, either by phosphorylation or dephosphorylation, might be responsible for altered B0AT1 protein transcription. Mast cells also release newly formed eicosanoids such as leukotriene C4, prostaglandin (PG)D2, and leukotriene B4. Of these, PGD2 has been found to play a major role in the progression of intestinal inflammation and has been shown to activate G-protein coupled receptors DP1 and DP2 resulting in the activation of adenylate cyclase and subsequent increase in intracellular cAMP levels and subsequent activation of the cAMP mediated protein kinase A (PKA) pathway [
33,
34]. The PKA pathway might regulate B0AT1 in the inflamed intestine by altering the phosphorylation status of transcription factors that regulate B0AT1 transcription. Similarly, PKA mediated direct phosphorylation of SN2 protein might be responsible for its altered affinity during inflammation. Previously, our laboratory has shown that an immune inflammatory mediator such as Leukotriene D4 alters the affinity of a different BBM amino acid co-transporter, specifically, Na-alanine co-transporter, ASCT1, and that this alteration was due to secondary changes in ASCT1 phosphorylation levels mediated by protein kinase C pathway [
29,
35].
In the present study, since mast cell stabilization reversed the increased affinity of SN2/SNAT5, as well as the inhibition of B0AT1 in the chronically inflamed rabbit intestine, it is reasonable to postulate that mast cell mediators function as an upstream common immune modulator of Na-glutamine co-transport alterations. Future studies are in order to determine the downstream immune inflammatory pathway/s and the specific inflammatory mediator/s that might be responsible for the regulation of Na-glutamine co-transporters B0AT1 and SN2 during intestinal inflammation.