Acute regulation of multidrug resistance-associated protein 2 localization and activity by cAMP and estradiol-17β-d-glucuronide in rat intestine and Caco-2 cells

Abstract

Multidrug resistance-associated protein 2 (MRP2) is an ATP-dependent transporter expressed at the brush border membrane of the enterocyte that confers protection against absorption of toxicants from foods or bile. Acute, short-term regulation of intestinal MRP2 activity involving changes in its apical membrane localization was poorly explored. We evaluated the effects of dibutyryl-cAMP (db-cAMP), a permeable analog of cAMP, and estradiol-17β-D-glucuronide (E17G), an endogenous derivative of estradiol, on MRP2 localization and activity using isolated rat intestinal sacs and Caco-2 cells, a model of human intestinal epithelium. Changes in MRP2 localization were studied by Western blotting of plasma membrane (PM) vs. intracellular membrane (IM) fractions in both experimental models, and additionally, by confocal microscopy in Caco-2 cells. After 30 min of exposure, db-cAMP-stimulated sorting of MRP2 from IM to PM both in rat jejunum and Caco-2 cells at 10 and 100 µM concentrations, respectively, with increased excretion of the model substrate 2,4-dinitrophenyl-S-glutathione. In contrast, E17G (400 µM) induced internalization of MRP2 together with impairment of transport activity. Confocal microscopy analysis performed in Caco-2 cells confirmed Western blot results. In the particular case of E17G, MRP2 exhibited an unusual pattern of staining compatible with endocytic vesiculation. Use of selective inhibitors demonstrated the participation of cAMP-dependent protein kinase and classic calcium-dependent protein kinase C in db-cAMP and E17G effects, respectively. We conclude that localization of MRP2 in intestine may be subjected to a dynamic equilibrium between plasma membrane and intracellular domains, thus allowing for rapid regulation of MRP2 function.

Intraluminal nutrients acutely strengthen rat intestinal MRP2 barrier function by a glucagon‐like peptide‐2‐mediated mechanism

“…We also found that PKA participation is necessary to explain, at least in part, modulation of MRP2 activity by adenosine (Figure 3). These findings agree with previous evidence in hepatic and intestinal models demonstrating the ability of cAMP/PKA axis to acutely increase MRP2 activity 22,51 . However, it is important to note that cAMP increase not only leads to PKA activation, but can also increase the activity of other kinases such as exchange protein directly activated by cAMP, phosphoinositide 3‐kinase or p38 mitogen‐activated protein kinases, all of them also able to acutely modulate MRP2 localization in hepatic models 51 .…”

“…Thus, Caco‐2 cell monolayers grown on permeable supports constitute an optimal in vitro model system for drug transport studies 40 . In addition, using confocal microscopy and membrane fractionation followed by immunoblotting studies, rapid changes in MRP2 localization were already confirmed in these cells 22 . In the current work, we demonstrated that acute treatment with adenosine led to a significant increase in MRP2 activity (Figure 3B) which was associated with apical insertion of the transporter (Figure 4).…”

“…In this study, we provide evidence supporting a novel physiological mechanism aimed at acutely regulating intestinal MRP2 activity in response to food intake, hence protecting the organism against the absorption of dietary toxicants. After our recent proof‐of‐concept article demonstrating the short‐term regulation of intestinal MRP2, 22 we focused on the development of an in vivo model with preserved irrigation and innervation that allows for detection of changes in MRP2 activity and localization in response to intraluminal administration of nutrients (section 4.2.2). This model was validated using the proinsertor agent dbcAMP (Figure 1A), intravenously administered at a dose known to be effective in modulating MRP2 in another highly perfused organ such as the liver 29 .…”

“…Recently, we have reported that intestinal MRP2 localization may be subjected to a dynamic equilibrium between BBM and intracellular domains, thus allowing for rapid regulation of MRP2 function 22 . This acute and bidirectional translocation was demonstrated for both rat and human orthologues using dibutyryl cAMP and estradiol‐17β‐D‐glucuronide.…”

“…The three main experimental findings described above can be summarized and sequentially linked as follows: (1) after intake of certain nutrients (such as oleic acid or glucose) GLP‐2 is released from L cells 23 ; (2) GLP‐2 triggers an increase in cAMP levels of rat enterocytes 21 ; (3) the increase in intracellular cAMP levels stimulates insertion of MRP2 into BBM with consequent impact on MRP2 transport activity 22 . This sequence of events, although possible, needs experimental demonstration and constitutes our current working hypothesis.…”