Activation of the calcium sensing receptor attenuates TRPV6-dependent intestinal calcium absorption

Abstract

Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+ sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxy vitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium, however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a non-functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co-expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.

Keywords: Calcium; Calcium channels; Gastroenterology; Nephrology.

Figure 1. Relative intestinal mRNA expression of transcellular Ca²⁺ transport mediators under altered extracellular Ca²⁺ conditions.

(A–C) Relative mRNA expression of transcellular Ca²⁺ transport mediators TRPV6 (Trpv6), CABP9K (S100g), NCX1 (Slc8a1), or PMCA1b (Atp2b1), normalized to 18S rRNA expression in mice on high-, normal- (Con), or low-Ca²⁺ diet for 21 days (n = 7 for each diet). (D–F) Relative mRNA expression in animals treated with 1,25-[OH]₂ D₃ (VD) or vehicle (Veh) (n = 8 for each). (G–I) Relative mRNA expression in animals treated with cinacalcet (Cin) or control (Veh) diet (n = 8 for each). All data are presented as the mean ± SEM, normalized to the mice on the normal/control diet. Asterisks indicate a statistically significant difference from the normal/control mice by 1-way ANOVA (all genes in A and Slc8a1 and Atp2b1 in B and C), Brown-Forsythe test (S100g in B), Kruskal-Wallis test (Trpv6 in B and C), or Student’s unpaired t tests (D–I); *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Protocol used to measure unidirectional Ca²⁺ fluxes across intestinal preparations.

The transepithelial voltage across tissue preparations (y axis) was clamped to 0 mV for the duration of the experiment (x axis). The voltage spikes along the x axis correspond to 2-mV pulses applied and used to determine the transepithelial resistance (TER). We added 0.1 μM tetrodotoxin (TTX) basolaterally first and allowed the resulting short-circuit current to stabilize. At time 0, the solutions were exchanged for fresh ones with 1 side spiked with ⁴⁵Ca²⁺. Asterisks indicate the time points when samples were taken for radioactivity measurements. Two gray horizontal lines represent 15-minute time intervals, where unidirectional ⁴⁵Ca²⁺ flux was calculated for each condition. We added 10 μM forskolin at the end of the experiment to confirm tissue viability.

Figure 3. The effect of altering extracellular Ca²⁺ on Ca²⁺ fluxes across mouse proximal colon.

(A) An example of the short-circuit current (I_SC) recorded throughout protocol 1. TTX was added and I_SC allowed to stabilize. The I_SC spikes occurred in response to 2-mV pulses. At the second arrow, solutions were exchanged, with 1 side only containing ⁴⁵Ca²⁺. The tissue was deemed viable if the I_SC increased more than 3 times with forskolin administration at the end of the experiment. (B) Changes in the net Ca²⁺ flux (net J_Ca²⁺) between condition A, pretreatment (Pre Rx), and condition B, vehicle (ethanol) or 10 μM cinacalcet (n = 6 each treatment). (C) The change in net J_Ca²⁺ between condition A, high Ca²⁺ (2.5 mM), and condition B, low-Ca²⁺ (0.5 mM), or the converse (n = 6 each). Raw values are presented; asterisks indicate a statistical difference between the conditions (Student’s paired t tests; *P < 0.05, and **P < 0.01).

Figure 4. Expression of the CaSR and effect of apical or basolateral CaSR activation.

(A) Relative mRNA expression of the CaSR throughout mouse intestine (n = 12), normalized to duodenum. (B and C) Changes in the net J_Ca²⁺ in the proximal colon of wild-type mice between condition A, pretreatment, and condition B, apical or basolateral 10 μM cinacalcet application (n = 7 each application in B; n = 6 in C). Raw values are presented; asterisks indicate a statistical difference between the conditions (Student’s paired t tests; *P < 0.05).

Figure 5. Effect of extracellular Ca²⁺ on Ca²⁺ fluxes across proximal colon from TRPV6^WT/WT or TRPV6^D541A/D541A mice.

(A) Change in net J_Ca²⁺ between condition A, pretreatment, and condition B, basolateral 10 μM cinacalcet application (n = 6 each). (B) Change in net J_Ca²⁺ between condition A, high Ca²⁺ (2.5 mM), and condition B, low Ca²⁺ (0.5 mM) (n = 6 each). Raw values are presented; asterisks indicate a statistical difference between conditions (Student’s paired t test for within genotype comparisons or unpaired t tests for between genotype comparison; *P < 0.05, and **P < 0.01).

Figure 6. Effect of phospholipase C inhibition on CaSR-mediated inhibition of Ca²⁺ absorption in in vitro and ex vivo.

(A) Effect of CaSR activation on Ca²⁺-induced currents (I_Ca) in TRPV6 expressing oocytes in the presence and absence of cinacalcet and/or U73122, a phospholipase C (PLC) inhibitor (n = 6 each). Mean I_Ca values obtained from TRPV6- and CaSR-expressing oocytes were normalized to vehicle I_Ca values from TRPV6-expressing oocytes ± SEM; asterisks indicate a statistically significant difference between the conditions (multiple-comparisons Kruskal-Wallis test; *P < 0.05; **P < 0.01). (B) Effect of cinacalcet and/or U73122 on the plasma membrane expression of TRPV6 and CaSR in oocytes determined by immunoblot. As a loading control, β-actin was blotted (bottom). (C) Quantification of surface TRPV6 expression, normalized to total TRPV6 (n = 3 each). (D) Effect of basolateral cinacalcet (10 μM) and vehicle (DMSO) or PLC inhibitor U73122 (10 μM) on mouse proximal colon (n = 6 each). Raw values are presented; asterisks indicate a statistical difference between the conditions (Student’s paired t tests; *P < 0.05).

Figure 7. Proposed model of CaSR-mediated inhibition of Ca²⁺ absorption.

(A) Increased plasma Ca²⁺ is sensed by the CaSR expressed in kidneys, bone, intestine, and parathyroid glands. In response, the kidneys decrease Ca²⁺ reabsorption, there is decreased bone resorption, and parathyroid hormone (PTH) secretion is decreased. We show herein that intestinal Ca²⁺ absorption is inhibited. In concert, this reduces plasma Ca²⁺. Importantly, PTH acts to increase plasma Ca²⁺ by increasing bone Ca²⁺ resorption, renal tubular reabsorption, and indirectly by increasing intestinal Ca²⁺ absorption by increasing 1,25-[OH]₂ D₃ synthesis in the kidneys. (B) High plasma Ca²⁺ is detected by the intestinal epithelial basolateral CaSR, which inhibits TRPV6-mediated transcellular Ca²⁺ transport via PLC.