Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein–coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1–secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca2+. In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca2+ response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber–mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.

Intestinal calcium (Ca²⁺) absorption occurs via paracellular and transcellular pathways. Although the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca²⁺ uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na⁺ fluxes. Consequently, we hypothesized that Na⁺ absorption mediates Ca²⁺ flux. NHE3 is central to intestinal Na⁺ absorption. NHE3 knockout mice (NHE3-/-) display impaired intestinal Na⁺, water, and Ca²⁺ absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca²⁺ absorption across different segments of wild-type mouse intestine. The cecum was the only segment with net Ca²⁺ absorption. Quantitative RT-PCR measurements revealed cecal expression of all genes implicated in intestinal Ca²⁺ absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 μM 5-(N-ethyl-N-isopropyl) amiloride decreased luminal-to-serosal and increased serosal-to-luminal Ca²⁺ flux. NHE3-/- mice had a >60% decrease in luminal-to-serosal Ca²⁺ flux. Ussing chambers experiments under altered voltage clamps (-25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca²⁺ absorption in NHE3-/- mice relative to wild-type animals. Consistent with this, cecal Trpv6 expression was diminished in NHE3-/- mice. Together these results implicate NHE3 in intestinal Ca(2+) absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na⁺ and water absorption.

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Calcium (Ca(2+)) is a key constituent in a myriad of physiological processes from intracellular signalling to the mineralization of bone. As a consequence, Ca(2+) is maintained within narrow limits when circulating in plasma. This is accomplished via regulated interplay between intestinal absorption, renal tubular reabsorption, and exchange with bone. Many studies have focused on the highly regulated active transcellular transport pathways for Ca(2+) from the duodenum of the intestine and the distal nephron of the kidney. However, comparatively little work has examined the molecular constituents creating the paracellular shunt across intestinal and renal epithelium, the transport pathway responsible for the majority of transepithelial Ca(2+) flux. More specifically, passive paracellular Ca(2+) absorption occurs across the majority of the intestine in addition to the renal proximal tubule and thick ascending limb of Henle's loop. Importantly, recent studies demonstrated that Ca(2+) transport through the paracellular shunt is significantly regulated. Therefore, we have summarized the evidence for different modes of paracellular Ca(2+) flux across renal and intestinal epithelia and highlighted recent molecular insights into both the mechanism of secondarily active paracellular Ca(2+) movement and the identity of claudins that permit the passage of Ca(2+) through the tight junction of these epithelia.