Edwin C. Y. Chow

Electrophysiologic control of motility in the human colon

Characteristics of electrical activities, and the relationship between electrical and motor activities, were studied in circular and longitudinal (taenia) muscle of the human colon that was obtained from 21 individuals. Recordings were obtained with suction electrodes, the sucrose-gap method, and microelectrodes. The circular muscle electrical activity consisted of oscillatory activity of relatively low amplitude, with a frequency range from 4.5 to 60 cycle/min. Spiking activity was present on most oscillations. Contractile activity was associated with individual oscillations at frequencies below 12 cycle/min. Contractions related to periods of oscillations at frequencies above 12 cycle/min showed summation resulting in prolonged contractions. In these periods, oscillations were either of relatively high amplitude, or had superimposed spiking activity. Longitudinal muscle activity consisted of slow electrical oscillations at frequencies between 24 and 36 cycle/min with spiking activity superimposed on most oscillations. Contractions were related to bursts of such activity. These findings provide the electrophysiologic basis for short and prolonged phasic contractions and for sustained contractions of the human colon muscle layers. Activities in both muscle layers were myogenic in nature, were very sensitive to stretch, and could be initiated or modulated by nervous activity.

Comparison of effects of VDR versus PXR, FXR and GR ligands on the regulation of CYP3A isozymes in rat and human intestine and liver

In this study, we compared the regulation of CYP3A isozymes by the vitamin D receptor (VDR) ligand 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) against ligands of the pregnane X receptor (PXR), the glucocorticoid receptor (GR) and the farnesoid X receptor (FXR) in precision-cut tissue slices of the rat jejunum, ileum, colon and liver, and human ileum and liver. In the rat, 1,25(OH)(2)D(3) strongly induced CYP3A1 mRNA, quantified by qRT-PCR, along the entire length of the intestine, induced CYP3A2 only in ileum but had no effect on CYP3A9. In contrast, the PXR/GR ligand, dexamethasone (DEX), the PXR ligand, pregnenolone-16 alpha carbonitrile (PCN), and the FXR ligand, chenodeoxycholic acid (CDCA), but not the GR ligand, budesonide (BUD), induced CYP3A1 only in the ileum, none of them influenced CYP3A2 expression, and PCN, DEX and BUD but not CDCA induced CYP3A9 in jejunum, ileum and colon. In rat liver, CYP3A1, CYP3A2 and CYP3A9 mRNA expression was unaffected by 1,25(OH)(2)D(3), whereas CDCA decreased the mRNA of all CYP3A isozymes; PCN induced CYP3A1 and CYP3A9, BUD induced CYP3A9, and DEX induced all three CYP3A isozymes. In human ileum and liver, 1,25(OH)(2)D(3) and DEX induced CYP3A4 expression, whereas CDCA induced CYP3A4 expression in liver only. In conclusion, the regulation of rat CYP3A isozymes by VDR, PXR, FXR and GR ligands differed for different segments of the rat and human intestine and liver, and the changes did not parallel expression levels of the nuclear receptors.

1Alpha,25-dihydroxyvitamin D3 up-regulates P-glycoprotein via the vitamin D receptor and not farnesoid X receptor in both fxr(-/-) and fxr(+/+) mice and increased renal and brain efflux of digoxin in mice in vivo.

Secondary farnesoid X receptor (FXR) effects, in addition to vitamin D receptor (VDR) effects, were observed in the rat liver after treatment with 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], the natural ligand of VDR, caused by increased bile acid absorption as a consequence of apical sodium-dependent bile acid transporter induction. To investigate whether the increased multidrug resistance protein 1 (Mdr1)/P-glycoprotein (P-gp) expression in the rat liver and kidney was caused by the VDR and not the FXR, we examined changes in Mdr1/P-gp expression in fxr(+/+) and fxr(−/−) mice after intraperitoneal dosing of vehicle versus 1,25(OH)2D3 (0 or 2.5 μg/kg every other day for 8 days). Renal and brain levels of Mdr1 mRNA and P-gp protein were significantly increased in both fxr(+/+) and fxr(−/−) mice treated with 1,25(OH)2D3, confirming that Mdr1/P-gp induction occurred independently of the FXR. Increased P-gp function was evident in 1,25(OH)2D3-treated fxr(+/+) mice given intravenous bolus doses of the P-gp probe, [3H]digoxin (0.1 mg/kg). Decreased blood (24%) and brain (29%) exposure, estimated as reduced areas under the curve, caused by increased renal (74%) and total body (34%) clearances of digoxin, were observed in treated mice. These events were predicted by physiologically based pharmacokinetic modeling that showed increased renal secretory intrinsic clearance (3.45-fold) and brain efflux intrinsic clearance (1.47-fold) in the 1,25(OH)2D3-treated mouse, trends that correlated well with increases in P-gp protein expression in tissues. The clearance changes were less apparent because of the high degree of renal reabsorption of digoxin. The observations suggest an important role of the VDR in the regulation of P-gp in the renal and brain disposition of P-gp substrates.

1 alpha,25-Dihydroxyvitamin D-3 Triggered Vitamin D Receptor and Farnesoid X Receptor-like Effects in Rat Intestine and Liver In Vivo

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3), a natural ligand of the vitamin D receptor (VDR), was found to increase the rat ileal Asbt and bile acid absorption. The effects of VDR, whose expression is low in liver, on hepatic transporters and enzymes are unknown. Protein and mRNA levels of target genes in the small intestine, colon and liver after intraperitoneal dosing of 1,25(OH)2D3 (0–2.56 nmol/kg/day for 4 days) to the rat were determined by Western blotting and qPCR, respectively. The 1,25(OH)2D3 treatment increased total Cyp3a protein and Cyp3a1 mRNA expressions in the proximal small intestine, and the short heterodimer partner (SHP), the fibroblast growth factor 15 (FGF15), organic solute transporter (Ostα and Ostβ) mRNA and Asbt protein expressions in the ileum. About 50% higher portal bile acid concentration (65.1±14.9 vs 41.9±7.8 µm, p<0.05) and elevated expressions of the hepatic farnesoid X receptor (FXR) and SHP mRNA resulted with 1,25(OH)2D3 treatment. Increased Bsep and Ostα mRNA expressions in liver and a>50% reduction in the Cyp7a1 protein level (p<0.05) and cholesterol metabolism in rat liver microsomes (p=0.002), likely consequences of the bile acid‐FXR‐SHP cascade and activation of the signaling pathway for Cyp7a1 inhibition by FGF15, were found. Increased hepatic multidrug resistance‐associated protein (Mrp3) and multidrug resistance protein 1a (Mdr1a) mRNA and P‐gp protein were also observed. It was concluded that the changes in hepatic transporters and enzymes in the rat were indirect, secondary effects of the liver FXR‐SHP cascade due to increased intestinal absorption of bile acids and elevated levels of FGF15, events that led to the activation of FXR. Copyright

1α,25-Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells.

Induction of the multidrug resistance protein 1 (MDR1)/P‐glycoprotein (P‐gp) by the vitamin D receptor (VDR) was investigated in isolated rat brain capillaries and rat (RBE4) and human (hCMEC/D3) brain microvessel endothelial cell lines. Incubation of isolated rat brain capillaries with 10 nM of the VDR ligand, 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3] for 4 h increased P‐gp protein expression fourfold. Incubation with 1,25(OH)2D3 for 4 or 24 h increased P‐gp transport activity (specific luminal accumulation of NBD‐CSA, the fluorescent P‐gp substrate) by 25–30%. In RBE4 cells, Mdr1b mRNA was induced in a concentration‐dependent manner by exposure to 1,25(OH)2D3. Concomitantly, P‐gp protein expression increased 2.5‐fold and was accompanied by a 20–35% reduction in cellular accumulation of the P‐gp substrates, rhodamine 6G (R6G), and HiLyte Fluor 488‐labeled human amyloid beta 1‐42 (hAβ42). In hCMEC/D3 cells, a 3 day exposure to 100 nM 1,25(OH)2D3 increased MDR1 mRNA expression (40%) and P‐gp protein (threefold); cellular accumulation of R6G and hAβ42 was reduced by 30%. Thus, VDR activation up‐regulates Mdr1/MDR1 and P‐gp protein in isolated rat brain capillaries and rodent and human brain microvascular endothelia, implicating a role for VDR in increasing the brain clearance of P‐gp substrates, including hAβ42, a plaque‐forming precursor in Alzheimers disease.

PBPK Modeling of Intestinal and Liver Enzymes and Transporters in Drug Absorption and Sequential Metabolism

Experimental strategies have long been applied for in vitro or in vivo evaluation of the effect of transporters and/or enzymes on the bioavailability. However, the lack of specific inhibitors or inducers of transporters and enzymes and the multiplicity of nuclear receptors in gene regulation and cross talk have led to compromised assessment of these effects in vivo. These and other causes have resulted in confusion and controversy in transporter-enzyme interplay. In this review, physiologically-based pharmacokinetic (PBPK) intestinal and liver models are utilized to predict the contributions of enzymes and transporters on intestinal availability (F(I)) and hepatic availability (F(H)), with the aim to fully understand the impact of these variables on bioavailability (F(sys)) in vivo. We emphasize the often overlooked impact of influx and efflux clearances, and apply the PBPK models and their solutions to examine individual organ clearances of the intestine and the liver. In order to accurately predict oral bioavailability, these organ models are incorporated into the whole body PBPK model, and additional complicated scenarios such as segmental differences and zonal heterogeneity of transporters and enzymes in the intestine and liver and segregated blood flow patterns of the intestine are further discussed. The sequential metabolism of a drug to form primary and secondary metabolites in the first-pass organs is considered in PBPK modeling, revealing that the segregated flow model (SFM) of the intestine is more appropriate than the traditional PBPK intestinal model (TM). Examples are included to highlight the potential application of these PBPK models on the quantitative prediction of bioavailability.

Effects of 1 alpha,25-Dihydroxyvitamin D-3 on Transporters and Enzymes of the Rat Intestine and Kidney In Vivo

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR‐rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill‐defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)2D3 treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)2D3 treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2‐ to 20‐fold) in 1,25(OH)2D3‐treated rats, and a 28‐fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal‐specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ostα, and Mrp4, and protein levels of PepT1 and Oat1 were decreased in a dose‐dependent manner, and the ∼50% concomitant reduction in FXR, SHP, HNF‐1α and HNF‐4α mRNA expression suggests the possibility of cross‐talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)2D3 changes are tissue‐specific, differing between the intestine and kidney which are VDR‐rich organs. Copyright

Temporal changes in tissue 1α,25-dihydroxyvitamin D3, vitamin D receptor target genes, and calcium and PTH levels after 1,25(OH)2D3 treatment in mice

The vitamin D receptor (VDR) maintains a balance of plasma calcium and 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], its natural active ligand, by directly regulating the calcium ion channel (TRPV6) and degradation enzyme (CYP24A1), and indirectly regulating the parathyroid hormone (PTH) for feedback regulation of the synthetic enzyme CYP27B1. Studies that examined the intricate relationships between plasma and tissue 1,25(OH)2D3 levels and changes in VDR target genes and plasma calcium and PTH are virtually nonexistent. In this study, we investigated temporal correlations between tissue 1,25(OH)2D3 concentrations and VDR target genes in ileum and kidney and plasma calcium and PTH concentrations in response to 1,25(OH)2D3 treatment in mice (2.5 μg/kg ip, singly or q2d × 4). After a single ip dose, plasma 1,25(OH)2D3 peaked at ∼0.5 h and then decayed biexponentially, falling below basal levels after 24 h and then returning to baseline after 8 days. Upon repetitive ip dosing, plasma, ileal, renal, and bone 1,25(OH)2D3 concentrations rose and decayed in unison. Temporal profiles showed increased expressions of ileal Cyp24a1 and renal Cyp24a1, Mdr1/P-gp, and VDR but decreased renal Cyp27b1 mRNA after a time delay in VDR activation. Increased plasma calcium and attenuated PTH levels and increased ileal and renal Trpv6 expression paralleled the changes in tissue 1,25(OH)2D3 concentrations. Gene changes in the kidney were more sustained than those in intestine, but the magnitudes of change for Cyp24a1 and Trpv6 were lower than those in intestine. The data revealed that 1,25(OH)2D3 equilibrates with tissues rapidly, and VDR target genes respond quickly to exogenously administered 1,25(OH)2D3.

Expression and Regulation of the Bile Acid Transporter, OST alpha-OST beta in Rat and Human Intestine and Liver

The regulation of the OSTα and OSTβ expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue slices. The gradient of protein and mRNA expression in segments of the intestine for rOSTα and rOSTβ paralleled that of rASBT. OSTα and OSTβ mRNA expression, quantified by qRT‐PCR, in rat jejunum, ileum, colon and liver, and in human ileum and liver was positively regulated by FXR and GR ligands. In contrast, the VDR ligand, 1,25(OH)2D3 decreased the expression of rOSTα‐rOSTβ in rat intestine, but had no effect on human ileum, and rat and human liver slices. Lithocholic acid (LCA) decreased the expression of rOSTα and rOSTβ in rat ileum but induced OSTα‐OSTβ expression in rat liver slices, and human ileum and liver slices. The PXR ligand, pregnenolone‐16α carbonitrile (PCN) had no effect. This study suggest that, apart from FXR ligands, the OSTα and OSTβ genes are also regulated by VDR and GR ligands and not by PXR ligands. This study show that VDR ligands exerted different effects on OSTα‐OSTβ in the rat and human intestine and liver compared with other nuclear receptors, FXR, PXR, and GR, pointing to species‐ and organ‐specific differences in the regulation of OSTα‐OSTβ genes. Copyright

A Catenary Model to Study Transport and Conjugation of Baicalein, a Bioactive Flavonoid, in the Caco-2 Cell Monolayer: Demonstration of Substrate Inhibition

The transport and metabolism of baicalein (Ba) was studied in vitro and in Caco-2 cells. Protein binding of Ba with Caco-2 lysate showed that Ba was bound to two classes of sites: a higher affinity, lower capacity site (KA1 = 27.6 ± 4.7 μM–1, n1 = 10.6 ± 0.6 nmol/mg) and lower affinity, higher capacity site (KA2 = 0.015 ± 0.0013 μM–1, n2 = 413 ± 21 nmol/mg). Incubation studies of Ba with Caco-2 lysate showed substrate inhibition of both glucuronidation and sulfation, with Km values of 0.14 ± 0.034 and 0.015 ± 0.0053 μM, and KI values of 6.75 ± 1.70 and 0.37 ± 0.16 μM, respectively. In the Caco-2 monolayer, Ba (8–47 μM) displayed good apparent permeabilities (Papp) across the membrane; Papp was found to be increased with elevated loading concentration in both the absorptive and secretory directions. However, the efflux ratio was less than unity, negating the involvement of apical efflux transporters. The concentration ratios of Ba sulfate (BS) and glucuronide (BG) decreased with increased loading Ba concentration, suggesting that BS and BG are apically excreted via transporters, likely breast cancer resistance protein and multidrug resistance-associated protein 2, respectively. Data fit to the catenary model, composed of basolateral, cellular, and apical compartments, showed a low cellular unbound fraction (0.0019 ± 0.00018), a high passive diffusion clearance (0.012 ± 0.00029 ml/min/mg), and substrate inhibition, with sulfation being more readily saturated and inhibited than glucuronidation, as evidenced by smaller Km value (0.35 ± 0.078 versus 1.95 ± 0.57 μM) and KI value (0.58 ± 0.20 versus 7.90 ± 1.10 μM); these patterns paralleled those observed in the lysate incubation studies. The results showed that the catenary model aptly predicts substrate inhibition kinetics and offers significant and mechanistic insight into the transport and atypical metabolism of drugs in the Caco-2 monolayer.