Narongrit Thongon

Prolactin stimulates transepithelial calcium transport and modulates paracellular permselectivity in Caco-2 monolayer: mediation by PKC and ROCK pathways

Prolactin (PRL) was previously demonstrated to rapidly enhance calcium absorption in rat duodenum and the intestine-like Caco-2 monolayer. However, its mechanism was not completely understood. Here, we investigated nongenomic effects of PRL on the transepithelial calcium transport and paracellular permselectivity in the Caco-2 monolayer by Ussing chamber technique. PRL increased the transcellular and paracellular calcium fluxes and paracellular calcium permeability within 60 min after exposure but decreased the transepithelial resistance of the monolayer. The effects of PRL could not be inhibited by RNA polymerase II inhibitor (5,6-dichloro-1-beta-D-ribobenzimidazole), confirming that PRL actions were nongenomic. Exposure to protein kinase C (PKC) or RhoA-associated coiled-coil forming kinase (ROCK) inhibitors (GF-109203X and Y-27632, respectively) abolished the stimulatory effect of PRL on transcellular calcium transport, whereas ROCK inhibitor, but not PKC inhibitor, diminished the PRL effect on paracellular calcium transport. Knockdown of the long isoform of PRL receptor (PRLR-L) also prevented the enhancement of calcium transport by PRL. In addition, PRL markedly increased paracellular sodium permeability and the permeability ratio of sodium to chloride, which are indicators of the paracellular charge-selective property and are known to be associated with the enhanced paracellular calcium transport. The permeability of other cations in the alkali metal series was also increased by PRL, and such increases were abolished by ROCK inhibitor. It could be concluded that PRL stimulated transepithelial calcium transport through PRLR-L and increased paracellular permeability to cations in the Caco-2 monolayer. These nongenomic actions of PRL were mediated by the PKC and ROCK signaling pathways.

Apical acidity decreases inhibitory effect of omeprazole on Mg 2+ absorption and claudin-7 and -12 expression in Caco-2 monolayers

Clinical studies reported hypomagnesaemia in long-term omeprazole usage that was probably due to intestinal Mg2+ wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg2+ transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg2+ absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg2+, but not Ca2+, transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg2+ transport-Mg2+ concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg2+ affinity of the paracellular channel. By continually monitoring the apical pH, we showed that omeprazole suppressed apical acid accumulation. Neomycin and spermine had no effect on passive Mg2+ transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg2+ transport in a calcium sensing receptor (CaSR)-independent manner. The results of western blot analysis showed that omeprazole significantly suppressed claudin (Cldn)-7 and -12, but not Cldn-2, expression in Caco-2 cells. By using apical solution of pH 5.5, 6.0, 6.5, and 7.0, we found that apical acidity markedly increased passive Mg2+ transport, Mg2+ affinity of the paracellular channel, and Cldn-7 and -12 expression in Caco-2 monolayers. Apical acidity abolished the inhibitory effect of omeprazole on passive Mg2+ transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg2+ absorption by luminal acidity-induced increase in Cldn-7 and -12 expression.

Enhancement of calcium transport in Caco-2 monolayer through PKCζ-dependent Cav1.3-mediated transcellular and rectifying paracellular pathways by prolactin

Previous investigations suggested that prolactin (PRL) stimulated the intestinal calcium absorption through phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK) signaling pathways. However, little was known regarding its detailed mechanisms for the stimulation of transcellular and voltage-dependent paracellular calcium transport. By using Ussing chamber technique, we found that the PRL-induced increase in the transcellular calcium flux and decrease in transepithelial resistance of intestinal-like Caco-2 monolayer were not abolished by inhibitors of gene transcription and protein biosynthesis. The PRL-stimulated transcellular calcium transport was completely inhibited by the L-type calcium channel blockers (nifedipine and verapamil) and plasma membrane Ca(2+)-ATPase (PMCA) inhibitor (trifluoperazine) as well as small interfering RNA targeting voltage-dependent L-type calcium channel Ca(v)1.3, but not TRPV6 or calbindin-D(9k). As demonstrated by (45)Ca uptake study, PI3K and PKC, but not ROCK, were essential for the PRL-enhanced apical calcium entry. In addition, PRL was unable to enhance the transcellular calcium transport after PKC(zeta) knockdown or exposure to inhibitors of PKC(zeta), but not of PKC(alpha), PKC(beta), PKC(epsilon), PKC(mu), or protein kinase A. Voltage-clamping experiments further showed that PRL markedly stimulated the voltage-dependent calcium transport and removed the paracellular rectification. Such PRL effects on paracellular transport were completely abolished by inhibitors of PI3K (LY-294002) and ROCK (Y-27632). It could be concluded that the PRL-stimulated transcellular calcium transport in Caco-2 monolayer was mediated by Ca(v)1.3 and PMCA, presumably through PI3K and PKC(zeta) pathways, while the enhanced voltage-dependent calcium transport occurred through PI3K and ROCK pathways.

The roles of acid-sensing ion channel 1a and ovarian cancer G protein-coupled receptor 1 on passive Mg2+ transport across intestinal epithelium-like Caco-2 monolayers

Intestinal passive Mg2+ absorption, which is vital for normal Mg2+ homeostasis, has been shown to be regulated by luminal proton. We aimed to study the regulatory role of intestinal acid sensors in paracellular passive Mg2+ transport. Omeprazole enhanced the expressions of acid-sensing ion channel 1a (ASIC1a), ovarian cancer G protein-coupled receptor 1 (OGR1), and transient receptor potential vanilloid 4 in Caco-2 cells. It also inhibited passive Mg2+ transport across Caco-2 monolayers. The expression and activation of OGR1 resulted in the stimulation of passive Mg2+ transport via phospholipase C- and protein kinase C-dependent pathways. ASIC1a activation, on the other hand, enhanced apical HCO3− secretion that led, at least in part, by a Ca2+-dependent pathway to an inhibition of paracellular Mg2+ absorption. Our results provided supporting evidence for the roles of OGR1 and ASIC1a in the regulation of intestinal passive Mg2+ absorption.

Omeprazole suppressed plasma magnesium level and duodenal magnesium absorption in male Sprague-Dawley rats

Hypomagnesemia is the most concerned side effect of proton pump inhibitors (PPIs) in chronic users. However, the mechanism of PPIs-induced systemic Mg2+ deficit is currently unclear. The present study aimed to elucidate the direct effect of short-term and long-term PPIs administrations on whole body Mg2+ homeostasis and duodenal Mg2+ absorption in rats. Mg2+ homeostasis was studied by determining the serum Mg2+ level, urine and fecal Mg2+ excretions, and bone and muscle Mg2+ contents. Duodenal Mg2+ absorption as well as paracellular charge selectivity were studied. Our result showed that gastric and duodenal pH markedly increased in omeprazole-treated rats. Omeprazole significantly suppressed plasma Mg2+ level, urinary Mg2+ excretion, and bone and muscle Mg2+ content. Thus, omeprazole induced systemic Mg2+ deficiency. By using Ussing chamber techniques, it was shown that omeprazole markedly suppressed duodenal Mg2+ channel-driven and Mg2+ channel-independent Mg2+ absorptions and cation selectivity. Inhibitors of mucosal HCO3− secretion significantly increased duodenal Mg2+ absorption in omeprazole-treated rats. We therefore hypothesized that secreted HCO3− in duodenum decreased luminal proton, this impeded duodenal Mg2+ absorption. Higher plasma total 25-OH vitamin D, diuresis, and urine PO43− were also demonstrated in hypomagnesemic rats. As a compensatory mechanism for systemic Mg2+ deficiency, the expressions of duodenal transient receptor potential melastatin 6 (TRPM6), cyclin M4 (CNNM4), claudin (Cldn)-2, Cldn-7, Cldn-12, and Cldn-15 proteins were enhanced in omeprazole-treated rats. Our findings support the potential role of duodenum on the regulation of Mg2+ homeostasis.

The inhibitory role of purinergic P2Y receptor on Mg2+ transport across intestinal epithelium-like Caco-2 monolayer

AbstractThe mechanism of proton pump inhibitors (PPIs) suppressing intestinal Mg2+ uptake is unknown. The present study aimed to investigate the role of purinergic P2Y receptors in the regulation of Mg2+ absorption in normal and omeprazole-treated intestinal epithelium-like Caco-2 monolayers. Omeprazole suppressed Mg2+ transport across Caco-2 monolayers. An agonist of the P2Y2 receptor, but not the P2Y4 or P2Y6 receptor, suppressed Mg2+ transport across control and omeprazole-treated monolayers. Omeprazole enhanced P2Y2 receptor expression in Caco-2 cells. Forskolin and P2Y2 receptor agonist markedly enhanced apical HCO3− secretion by control and omeprazole-treated monolayers. The P2Y2 receptor agonist suppressed Mg2+ transport and stimulated apical HCO3− secretion through the Gq-protein coupled-phospholipase C (PLC) dependent pathway. Antagonists of cystic fibrosis transmembrane conductance regulator (CFTR) and Na+-HCO3− cotransporter-1 (NBCe1) could nullify the inhibitory effect of P2Y2 receptor agonist on Mg2+ transport across control and omeprazole-treated Caco-2 monolayers. Our results propose an inhibitory role of P2Y2 on intestinal Mg2+ absorption.