Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis

Abstract

The only Na-nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na-nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2-week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used.

Results: Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation.

Keywords: Glutamine transport; SN2/SNAT5; leukotrienes; prostaglandins and crypt cells.

Figure 1

(A) Effect of ATK on SN2 in intact crypt cells. Na‐dependent glutamine uptake was significantly increased in crypt cells from the chronically inflamed intestine. Rabbits with chronic enteritis treated with ATK reversed NGcT stimulation. ATK had no effect on NGcT in crypt cells from the normal intestine. (B) Effect of ATK on Na⁺/K⁺‐ATPase. Na⁺/K⁺‐ATPase activity was significantly increased in the crypt cells during chronic enteritis and was reversed by ATK treatment. Na⁺/K⁺‐ATPase activity in ATK‐treated normal intestinal crypt cells remained unaffected.

Figure 2

Effect of ATK on SN2 in crypt cells BBMV. Na‐dependent glutamine uptake was significantly increased in crypt cell BBMV from the chronically inflamed intestine. This inhibition was reversed with in vivo ATK treatment. NGcT was not affected by ATK treatment in the normal intestine.

Figure 3

Kinetics of Na‐dependent glutamine uptake in villus BBMV. As the concentration of extravesicular glutamine was increased, the uptake of Na‐dependent glutamine also increased and subsequently became saturated in all conditions. The kinetic parameters derived from these data are shown in Table 1.

Figure 4

SN2 protein expression in crypt cell BBM. SN2 protein expression in crypt cell BBM from control, chronic enteritis and ATK‐treated rabbits. A representative Western blot of BBM SN2 protein is shown in the upper panel. In the lower panel, densitometric quantitation is shown. The relative protein expression levels of SN2 are unaffected among all the groups. Ezrin was used as internal control.

Figure 5

Effect of piroxicam on SN2 in crypt cells BBMV. Na‐dependent glutamine uptake was significantly increased in crypt cell BBMV from the chronically inflamed intestine. This stimulation was not reversed with in vivo piroxicam treatment. Na‐glutamine cotransport was not affected in the normal intestine treated with piroxicam. These data indicated that Na‐glutamine cotransport in crypt cells are likely not regulated by COX pathway metabolites in the chronically inflamed intestine.

Figure 6

Effect of MK886 on SN2 in crypt cells BBMV. Na‐dependent glutamine uptake was significantly increased in crypt cell BBMV from the chronically inflamed intestine. This stimulation was reversed by in vivo MK886 treatment. Na‐glutamine cotransport was not affected in the normal intestine treated with MK886. These data indicate that Na‐glutamine cotransport in crypt cells likely regulated by LOX pathway metabolites in the chronically inflamed intestine.

Figure 7

Kinetics of Na‐dependent glutamine uptake in crypt cell BBMV. As the concentration of extravesicular glutamine was increased, the uptake of Na‐dependent glutamine also increased and subsequently became saturated in all conditions. The kinetic parameters derived from these data are shown in Table 2.

Figure 8

SN2 protein expression in crypt cell BBM. SN2 protein expression in crypt cell BBM from control, chronic enteritis and MK886‐treated rabbits. A representative Western blot of BBM SN2 protein is shown in the upper panel. Ezrin was used as loading control. In the lower panel, densitometric quantitation is shown. The relative protein expression levels of SN2 are unaffected among all the groups.

Figure 9

AA‐mediated regulation of NGcT. Na‐dependent glutamine cotransporter SN2 in crypt cells is facilitated by AA pathway particularly through LOX pathway during chronic intestinal inflammation.