Celastrus paniculatus seed extract exhibits neuroprotective effects against MPP+­induced apoptotic cell death via GSK­3ß in a Parkinson's disease model.

Parkinson's disease (PD) is a common neurodegenerative disease induced by the death of dopaminergic neurons. Seed oil of Celastrus paniculatus (CP) Willd. has protective and antioxidant properties; however, to the best of our knowledge, no studies have analyzed the neuroprotective effect of CP seeds on PD. The present study aimed to investigate the neuroprotective effects and mechanism of CP seed extract (CPSE) using an in vitro 1-methyl-4-phenylpyridinium ion (MPP+)-induced PD model. The effect of CPSE on the expression levels of apoptotic marker proteins, such as Bcl-2 and its upstream pathway protein, glycogen synthase kinase-3ß (GSK-3ß), was investigated in human neuroblastoma SH-SY5Y cells. The effect of CPSE on the viability of SH-SY5Y cells was evaluated using MTT assay. To investigate the potential neuroprotective effect of CPSE, SH-SY5Y cells were treated with MPP+ to induce PD-associated cytotoxicity. SH-SY5Y cells were treated with 2 mM MPP+ before or after CPSE treatment to determine the protective effect of CPSE against MPP+-induced neurotoxicity using MTT, WST-1 and lactate dehydrogenase assays. Moreover, it was investigated whether CPSE could promote survival signals by regulating the protein expression levels of apoptotic markers (Bcl-2 and GSK-3ß) using western blotting. High concentrations and prolonged treatment of CPSE did not have any adverse effect on SH-SY5Y cell viability. Furthermore, MPP+-induced dopaminergic neuron damage was ameliorated by CPSE treatment. CPSE also showed anti-apoptotic activity by reversing the inhibitory effects of MPP+ on Bcl-2 expression. Moreover, CPSE abolished MPP+-induced decreases in phosphorylated-GSK-3ß (Ser9) expression. Taken together, the present findings suggested that CPSE may exert a neuroprotective effect in PD.

Current opinion on the regulation of small intestinal magnesium absorption.

Magnesium (Mg2+) has an important role in numerous biological functions, and Mg2+ deficiency is associated with several diseases. Therefore, adequate intestinal absorption of Mg2+ is vital for health. The small intestine was previously thought to absorb digested Mg2+ exclusively through an unregulated paracellular mechanism, which is responsible for approximately 90% of total Mg2+ absorption. Recent studies, however, have revealed that the duodenum, jejunum, and ileum absorb Mg2+ through both transcellular and paracellular routes. Several regulatory factors of small intestinal Mg2+ uptake also have been explored, e.g., parathyroid hormone, fibroblast growth factor-23, apical acidity, proton pump inhibitor, and pH-sensing channel and receptors. The mechanistic factors underlying proton pump inhibitor suppression of small intestinal Mg2+, such as magnesiotropic protein dysfunction, higher mucosal bicarbonate secretion, Paneth cell dysfunction, and intestinal inflammation, are currently being explored. The potential role of small intestinal microbiomes in Mg2+ absorption has also been proposed. In this article, we reviewed the current knowledge on the mechanisms and regulatory factors of small intestinal Mg2+ absorption.

Hippocampal synaptic dysfunction and spatial memory impairment in omeprazole-treated rats.

Although the association of prolonged use of proton pump inhibitors, such as omeprazole, with memory impairment has been reported more than two decades ago, its underlying molecular mechanism is yet to be determined. Thus, in this study, we aimed to determine the mechanisms underlying the effect of prolonged omeprazole treatment on hippocampal synaptic function and spatial memory in male rats. Adult rats were subcutaneously administered with omeprazole for 12 or 24 weeks. Spatial memory was assessed using the Morris water maze (MWM) test. We examined the hippocampal protein expression of synaptic plasticity proteins, including the AMPA receptor subunit GluA1, postsynaptic density-95 (PSD-95), and activity-regulated cytoskeleton-associated protein (Arc), and the hippocampal expression and localization of androgen receptor (AR). In the MWM test, the escape latency was found to be significantly higher, and the number of platform crossings and the time spent in the target quadrant were significantly lower in the rats treated with omeprazole compared to the control rats. Hypomagnesemia and lower bone and brain Mg2+ content were also detected in the omeprazole-treated groups compared with the control group. The expression of GluA1, PSD-95, and Arc in the hippocampus and the expression of AR in the dentate gyrus and CA1 of the hippocampus were significantly lower in the omeprazole-treated groups than in the control group. These results suggest that prolonged omeprazole treatment might lead to memory deficit by impairing glutamate receptor trafficking or synaptic anchoring. Hypomagnesemia and brain Mg2+ deficiency may be, at least in part, involved in omeprazole-induced memory impairment.

Mass spectrometric analysis of TRPM6 and TRPM7 from small intestine of omeprazole-induced hypomagnesemic rats.

Disruption of small intestinal Mg2+ absorption has been reported as the underlying mechanism of proton pump inhibitor-induced hypomagnesemia (PPIH); hence, this study evaluated the expression, localization, phosphorylation, and oxidation of transient receptor potential melastatin 6 (TRPM6) and TRPM7 in the small intestine of rats subjected to PPIH. The expression and localization of cyclin M4 (CNNM4) was also analyzed. We show that, compared to control rats, membrane expression of the TRPM6/7 heterodimer and TRPM7 was markedly lower in the duodenum and the jejunum of PPIH rats; in contrast, expression of membrane TRPM6 and CNNM4 was higher in these organs. Mass spectrometric analysis of TRPM6 demonstrated hyper-phosphorylation, especially T1851, and hyper-oxidation at M1755, both of which can suppress its channel permeability. Further, hypo-phosphorylation of S141 and the dimerization motif domain of TRPM6 in PPIH rats might be involved in lower TRPM6/7 heterodimer expression. Hypo-phosphorylation, especially at S138 and S1360 in TRPM7 from PPIH rats disrupted stability of TRPM7 at the cell membrane; hyper-oxidation of TRPM7 was also observed. These results help explain the mechanism underlying the disruption of small intestinal Mg2+ absorption in PPIH.

Fibroblast growth factor-23 and parathyroid hormone suppress small intestinal magnesium absorption.

In the present study, we examined the systemic and direct effects of parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23) on duodenal, jejunal, and ileal Mg2+ absorption. The rats were injected with FGF-23 or PTH for 5 h before collecting the duodenum, jejunum, and ileum for Mg2+ transport analysis in Ussing chambers. The duodenum, jejunum, and ileum were directly exposed to FGF-23, PTH, or FGF-23 plus PTH with or without cell signaling inhibitors for 150 min in Ussing chambers prior to performing the Mg2+ transport study. The small intestinal tissues were also subjected to western blot analyses for FGF receptor (FGFR), PTH receptor (PTHR), Klotho, transient receptor potential melastatin 6 (TRPM6), and cyclin as well as the cystathionine ß-synthase domain divalent metal cation transport mediator 4 (CNNM4) expression. The small intestine abundantly expressed FGFR and PTHR proteins, whereas, Klotho was not expressed in rat small intestine. Systemic PTH or FGF-23 injection significantly suppressed transcellular Mg2+ transport in the duodenum and jejunum. Direct FGF-23-, PTH-, or FGF-23 plus PTH exposure also suppressed transcellular Mg2+ absorption in the duodenum and jejunum. There was no additional inhibitory effect of PTH and FGF-23 on intestinal Mg2+ absorption. The inhibitory effect of PTH, FGF-23, or FGF-23 plus PTH was abolished by Gö 6850. Systemic PTH- or FGF-23-injection significantly decreased membranous TRPM6 expression, but increased cytosolic CNNM4 expression in the duodenum, jejunum, and ileum. In the present study, we propose a novel magnesiotropic action of PTH and FGF-23 by modulating small intestinal Mg2+ absorption.

Ultrastructural intestinal mucosa change after prolonged inhibition of gastric acid secretion by omeprazole in male rats.

Omeprazole is a potent inhibitor of gastric acid secretion. It was reported that omeprazole induced dramatic gastric mucosa morphologic changes from the resting state to the stimulated state. However, the effect of omeprazole administration on the ultrastructure and absorptive function of small intestines was largely unknown. Here, male Sprague-Dawley rats were daily treated with a single dose of omeprazole for 12 or 24 weeks. Ultrastructure intestinal mucosal change in duodenum, jejunum, and ileum was observed. We also determined small intestine inflammation, using intraepithelial lymphocytes activation. Finally, magnesium levels were measured in plasma, urine, feces, muscle, and bone to determine systemic magnesium balance. Omeprazole-treated rats had significantly decreased the width of tight junction, villous length, and absorptive area of duodenum, jejunum, and ileum compared to control rats. The small intestine of the omeprazole-treated group showed significantly higher intraepithelial lymphocytes activation levels compared with the control group. Lower secretory granules of Paneth cells at the base of the crypts were showed in omeprazole-treated rats. They also had significantly lower plasma, urinary, bone, and muscle Mg2+ contents indicating hypomagnesemia with systemic magnesium deficiency. In conclusion, prolonged omeprazole treatment-induced small intestinal inflammation and villous atrophy, which led to decrease small intestinal magnesium absorption in the condition of proton pump inhibitor-induced hypomagnesemia.

Effect of prolonged omeprazole administration on segmental intestinal Mg2+ absorption in male Sprague-Dawley rats.

BACKGROUND: The exact mechanism of proton pump inhibitors (PPIs)-induced hypomagnesemia (PPIH) is largely unknown. Previous studies proposed that PPIH is a consequence of intestinal Mg2+ malabsorption. However, the mechanism of PPIs-suppressed intestinal Mg2+ absorption is under debate. AIM: To investigate the effect of 12-wk and 24-wk omeprazole injection on the total, transcellular, and paracellular Mg2+ absorption in the duodenum, jejunum, ileum, and colon of male Sprague-Dawley rats. METHODS: The rats received 20 mg/kg∙d subcutaneous omeprazole injection for 12 or 24 wk. Plasma and urinary Mg2+, Ca2+, and PO4 3- levels were measured. The plasma concentrations of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23), epidermal growth factor (EGF), and insulin were also observed. The duodenum, jejunum, ileum, and colon of each rat were mounted onto individual modified Using chamber setups to study the rates of total, transcellular, and paracellular Mg2+ absorption simultaneously. The expression of transient receptor potential melastatin 6 (TRPM6) and cyclin M4 (CNNM4) in the entire intestinal tract was also measured. RESULTS: Single-dose omeprazole injection significantly increased the intraluminal pH of the stomach, duodenum, and jejunum. Omeprazole injection for 12 and 24 wk induced hypomagnesemia with reduced urinary Mg2+ excretion. The plasma Ca2+ was normal but the urinary Ca2+ excretion was reduced in rats with PPIH. The plasma and urinary PO4 3- levels increased in PPIH rats. The levels of 1α,25(OH)2D3 and FGF-23 increased, whereas that of plasma EGF decreased in the omeprazole-treated rats. The rates of the total, transcellular, and paracellular Mg2+ absorption was significantly lower in the duodenum, jejunum, ileum, and colon of the rats with PPIH than in those of the control rats. The percent suppression of Mg2+ absorption in the duodenum, jejunum, ileum, and colon of the rats with PPIH compared with the control rats was 81.86%, 70.59%, 69.45%, and 39.25%, respectively. Compared with the control rats, the rats with PPIH had significantly higher TRPM6 and CNNM4 expression levels throughout the intestinal tract. CONCLUSION: Intestinal Mg2+ malabsorption was observed throughout the intestinal tract of rats with PPIH. PPIs mainly suppressed small intestinal Mg2+ absorption. Omeprazole exerted no effect on the intraluminal acidic pH in the colon. Thus, the lowest percent suppression of total Mg2+ absorption was found in the colon. The expression levels of TRPM6 and CNNM4 increased, indicating the presence of a compensatory response to Mg2+ malabsorption in rats with PPIH. Therefore, the small intestine is an appropriate segment that should be modulated to counteract PPIH.

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

The 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.

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 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 Mg(2+) absorption, which is vital for normal Mg(2+) homeostasis, has been shown to be regulated by luminal proton. We aimed to study the regulatory role of intestinal acid sensors in paracellular passive Mg(2+) 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 Mg(2+) transport across Caco-2 monolayers. The expression and activation of OGR1 resulted in the stimulation of passive Mg(2+) 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 Ca(2+)-dependent pathway to an inhibition of paracellular Mg(2+) absorption. Our results provided supporting evidence for the roles of OGR1 and ASIC1a in the regulation of intestinal passive Mg(2+) absorption.

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 Mg(2+) wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg(2+) transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg(2+) absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg(2+), but not Ca(2+), transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg(2+) transport-Mg(2+) concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg(2+) 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 Mg(2+) transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg(2+) 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 Mg(2+) transport, Mg(2+) 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 Mg(2+) transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg(2+) absorption by luminal acidity-induced increase in Cldn-7 and -12 expression.

Omeprazole decreases magnesium transport across Caco-2 monolayers.

AIM: To elucidate the effect and underlying mechanisms of omeprazole action on Mg(2+) transport across the intestinal epithelium. METHODS: Caco-2 monolayers were cultured in various dose omeprazole-containing media for 14 or 21 d before being inserted into a modified Ussing chamber apparatus to investigate the bi-directional Mg(2+) transport and electrical parameters. Paracellular permeability of the monolayer was also observed by the dilution potential technique and a cation permeability study. An Arrhenius plot was performed to elucidate the activation energy of passive Mg(2+) transport across the Caco-2 monolayers. RESULTS: Both apical to basolateral and basolateral to apical passive Mg(2+) fluxes of omeprazole-treated epithelium were decreased in a dose- and time-dependent manner. Omeprazole also decreased the paracellular cation selectivity and changed the paracellular selective permeability profile of Caco-2 epithelium to Li(+), Na(+), K(+), Rb(+), and Cs(+) from series VII to series VI of the Eisenman sequence. The Arrhenius plot revealed the higher activation energy for passive Mg(2+) transport in omeprazole-treated epithelium than that of control epithelium, indicating that omeprazole affected the paracellular channel of Caco-2 epithelium in such a way that Mg(2+) movement was impeded. CONCLUSION: Omeprazole decreased paracellular cation permeability and increased the activation energy for passive Mg(2+) transport of Caco-2 monolayers that led to the suppression of passive Mg(2+) absorption.

Transepithelial calcium transport in prolactin-exposed intestine-like Caco-2 monolayer after combinatorial knockdown of TRPV5, TRPV6 and Ca(v)1.3.

The milk-producing hormone prolactin (PRL) increases the transcellular intestinal calcium absorption by enhancing apical calcium uptake through voltage-dependent L-type calcium channel (Ca(v)) 1.3. However, the redundancy of apical calcium channels raised the possibility that Ca(v)1.3 may operate with other channels, especially transient receptor potential vanilloid family calcium channels (TRPV) 5 or 6, in an interdependent manner. Herein, TRPV5 knockdown (KD), TRPV5/TRPV6, TRPV5/Ca(v)1.3, and TRPV6/Ca(v)1.3 double KD, and TRPV5/TRPV6/Ca(v)1.3 triple KD Caco-2 monolayers were generated by transfecting cells with small interfering RNAs (siRNA). siRNAs downregulated only the target mRNAs, and did not induce compensatory upregulation of the remaining channels. After exposure to 600 ng/mL PRL, the transcellular calcium transport was increased by ~2-fold in scrambled siRNA-treated, TRPV5 KD and TRPV5/TRPV6 KD monolayers, but not in TRPV5/Ca(v)1.3, TRPV6/Ca(v)1.3 and TRPV5/TRPV6/Ca(v)1.3 KD monolayers. The results suggested that Ca(v)1.3 was the sole apical channel responsible for the PRL-stimulated transcellular calcium transport in intestine-like Caco-2 monolayer.

Two-step stimulation of intestinal Ca(2+) absorption during lactation by long-term prolactin exposure and suckling-induced prolactin surge.

During pregnancy and lactation, the enhanced intestinal Ca(2+) absorption serves to provide Ca(2+) for fetal development and lactogenesis; however, the responsible hormone and its mechanisms remain elusive. We elucidated herein that prolactin (PRL) markedly stimulated the transcellular and paracellular Ca(2+) transport in the duodenum of pregnant and lactating rats as well as in Caco-2 monolayer in a two-step manner. Specifically, a long-term exposure to PRL in pregnancy and lactation induced an adaptation in duodenal cells at genomic levels by upregulating the expression of genes related to transcellular transport, e.g., TRPV5/6 and calbindin-D(9k), and the paracellular transport, e.g., claudin-3, thereby raising Ca(2+) absorption rate to a new "baseline" (Step 1). During suckling, PRL surge further increased Ca(2+) absorption to a higher level (Step 2) in a nongenomic manner to match Ca(2+) loss in milk. PRL-enhanced apical Ca(2+) uptake was responsible for the increased transcellular transport, whereas PRL-enhanced paracellular transport required claudin-15, which regulated epithelial cation selectivity and paracellular Ca(2+) movement. Such nongenomic PRL actions were mediated by phosphoinositide 3-kinase, protein kinase C, and RhoA-associated coiled-coil-forming kinase pathways. In conclusion, two-step stimulation of intestinal Ca(2+) absorption resulted from long-term PRL exposure, which upregulated Ca(2+) transporter genes to elevate the transport baseline, and the suckling-induced transient PRL surge, which further increased Ca(2+) transport to the maximal capacity. The present findings also suggested that Ca(2+) supplementation at 15-30 min prior to breastfeeding may best benefit the lactating mother, since more Ca(2+) could be absorbed as a result of the suckling-induced PRL surge.

Enhancement of calcium transport in Caco-2 monolayer through PKCzeta-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.

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.

Osteoblasts express claudins and tight junction-associated proteins.

Osteoblasts were previously reported to form tight junctions, which may play an important role in the regulation of ion transport across the epithelial-like bone membrane. However, the evidence for the presence of tight junction-associated proteins in osteoblasts is lacking. We therefore studied the expression of tight junction-associated genes in primary rat osteoblasts and bone tissues. Quantitative real-time PCR showed that osteoblasts expressed ZO-1, -2, -3, cingulin, occludin, claudin-1 to -12, -14 to -20, -22 and -23. By using western blot analyses of selected claudins, expression of claudin-5, -11, -14 and -15, but not claudin-3, were identified in osteoblasts. A confocal immunofluorescent study in undecalcified tibial sections confirmed that claudin-16 was localized on the trabecular surface, normally covered by osteoblasts and bone-lining cells. In addition, immunohistochemical studies in decalcified tibial sections demonstrated the expression of claudin-5, -11, -14, -15 and -16 in bone-lining cells (inactive osteoblasts). Primary osteoblasts cultured in the Snapwell for 19-26 days were found to form a monolayer with measurable transepithelial resistance of approximately 110-180 Omegacm(2), confirming the presence of barrier functions of the tight junction. It was concluded that osteoblasts expressed several tight junction-associated proteins, which possibly regulated ion transport across the bone membrane.

Prolactin-stimulated transepithelial calcium transport in duodenum and Caco-2 monolayer are mediated by the phosphoinositide 3-kinase pathway.

Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/ml. This response diminished after exposure to LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/ml. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY-294002 and wortmannin), but not by inhibitors of MEK (U-0126) or JAK2 (AG-490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride (P(Na)/P(Cl)) was determined. We found that PRL elevated the P(Na)/P(Cl) in both epithelia, and the effects were blocked by PI3K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the nongenomic PI3K-signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity.