Functional expression of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) mediate the cellular uptake of nucleosides and nucleobases across the plasma membrane and play important roles in the salvage pathways of nucleotide synthesis. However, information about nucleoside transport systems in the lung alveolar epithelial cells is limited. Therefore, in the present study, we examined the function and expression of nucleoside transporters using primary cultured alveolar type II cells and transdifferentiated type I-like cells. The uptake of uridine, a substrate for ENTs and CNTs, in type II and type I-like cells was time, temperature, and concentration dependent, and was inhibited by other nucleoside transporter substrates such as adenosine. Uridine uptake in both cells was insensitive to nanomolar concentrations of NBMPR, a potent ENT1 inhibitor, while it was inhibited by higher concentrations of NBMPR, suggesting that ENT2, but not ENT1, is involved in uridine uptake in these cells. Additionally, uridine uptake was higher in the presence of Na+ than in the absence of Na + and was partially inhibited by a CNT inhibitor phloridzin in these cells, suggesting that CNT is also involved in uridine uptake. In both cells, the mRNA expression of ENT1, ENT2, CNT2, and CNT3 was observed. Finally, the activity of uridine uptake was considerably higher in type II cells than in type I-like cells. In addition, the mRNA expression of ENT2, CNT2, and CNT3, but not ENT1, was lower in type I-like cells than in type II cells. These findings would help understand the functional roles of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Transport of ribavirin in human myelogenous leukemia cell line K562.

The anticancer effect of ribavirin, a purine nucleoside analogue, has been studied using cultured cancer cells such as the human myelogenous leukemia cell line K562. In order to exert its pharmacological effect, ribavirin has to enter cancer cells. However, there is little information concerning the transport mechanism of ribavirin into K562 cells. In this study, therefore, we examined the uptake mechanism of ribavirin in K562 cells. The uptake of ribavirin in K562 cells was time- and temperature-dependent, and was saturable with a Km value of 1.5 mM. Ribavirin uptake was inhibited by nucleosides such as adenosine and uridine, and by inhibitors of equilibrative nucleoside transporter 1 (ENT1) such as S-(4-nitrobenzyl)-6-thioinosine and dipyridamole in a concentration-dependent manner. In addition, the expression of ENT1 mRNA in K562 cells was confirmed by real-time PCR. On the other hand, Na+-dependence of ribavirin uptake was not observed, suggesting the involvement of ENT1, but not Na+-dependent concentrative nucleoside transporters, in ribavirin uptake in K562 cells. Treatment of K562 cells with sodium butyrate induced erythroid differentiation, but ribavirin uptake activity and sensitivity of the uptake to various inhibitors were not different between native and differentiated K562 cells. These results suggest that ribavirin uptake into K562 cells is mainly mediated by ENT1, which may have a pivotal role in anticancer effect of ribavirin.

Functional expression of breast cancer resistance protein and cholesterol effect in human erythrocyte membranes.

In human erythrocyte membranes, various influx and efflux transporters are functionally expressed. However, their transport characteristics and modulation under disease states are not fully understood. In this study, we first examined the expression and detailed transport characteristics of breast cancer resistance protein (BCRP), an efflux ABC transporter, using inside-out membrane vesicles (IOVs) prepared from human erythrocytes, and then studied the effect of membrane cholesterol on BCRP function. The expression of BCRP was confirmed by western blotting; most of them being homodimers. The uptake of lucifer yellow (LY), a fluorescent BCRP substrate, into IOVs was time-, temperature-, and ATP-dependent, and the concentration of ATP which induced half-maximal stimulation of LY uptake was calculated to be 0.39 mM. The uptake of LY by IOVs was saturable with a Km value of 166 µM, and was inhibited by various BCRP inhibitors and substrates, such as fumitremorgin C and mitoxantrone. When membrane cholesterol content was increased by treating IOVs with cholesteryl hemisuccinate, LY uptake decreased with increasing cholesterol content. These results suggest that transport activity of BCRP in human erythrocyte membranes may be suppressed under disease states, such as hypercholesterolemia, that increase membrane cholesterol content.

P-gp modulating effect of Azadirachta indica extract in multidrug-resistant cancer cell lines.

The extract of Azadirachta indica, commonly known as neem, has found extensive use in traditional medicine for treating various human diseases. In this study, the effect of the 50% ethanol extract of A. indica (AI01) on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) was examined using MDR cell lines, specifically paclitaxel-resistant HepG2 (PR-HepG2) and doxorubicin (DOX)-resistant (DR) colon-26 cells. 96-h treatment of the two cell lines with AI01 (30 µg/mL) showed no effect on the expression of P-gp mRNA (human MDR1 and mouse mdr1b) and protein, while AI01 increased the accumulation of rhodamine 123, a P-gp substrate, in both PR-HepG2 and DR-colon-26 cells. The cytotoxic effects of 48-h treatment with AI01 on the viability of PR-HepG2 and DR-colon-26 cells were not observed. Therefore, 30 µg/mL AI01 may have no cytotoxic and P-gp-inducing effects. Finally, AI01 potentiated the sensitivity of PR-HepG2 and DR-colon-26 cell lines to DOX by 8.6- and 15.3-fold, respectively. These findings suggest that A. indica may be a promising source for a new class of P-gp modulators without cytotoxic/P-gp induction effects.

Effect of Curcuma comosa extracts on the functions of peptide transporter and P-glycoprotein in intestinal epithelial cells.

Curcuma comosa has been widely used as a herbal medicine in Thailand; however, it remains unclear whether C. comosa influences the absorption of drugs that are substrates for the transporters in the small intestine. In this study, we investigated the effect of C. comosa extracts on the functioning of peptide transporter 1 (PEPT1), an influx transporter, and P-glycoprotein (P-gp), an efflux transporter, in Caco-2 cells and rat intestine. In Caco-2 cells, the ethanolic extract of C. comosa (CCE) lowered the uptake of glycylsarcosine (Gly-Sar), a PEPT1 substrate, while it enhanced the uptake of rhodamine 123 (Rho123), a P-gp substrate, in a concentrationdependent manner. In addition, CCE inhibited apical-to-basal transport of Gly-Sar and basal-to-apical transport of Rho123. Furthermore, the absorption of cephalexin, another PEPT1 substrate, and the exsorption of Rho123 across the rat intestine were inhibited by CCE. Conversely, CCW, the hot water extract of C. comosa, suppresses the function of PEPT1 but not of P-gp in Caco-2 cells. These results suggest that C. comosa used as a herbal medicine in Thailand may affect the intestinal absorption of certain drugs.

Modulation of P-glycoprotein function and multidrug resistance in cancer cells by Thai plant extracts.

The effects of ethanol extracts from Thai plants belonging to the families of Annonaceae, Rutaceae, and Zingiberaceae on P-glycoprotein (P-gp) function and multidrug resistance were examined in paclitaxel-resistant HepG2 (PR-HepG2) cells. All the extracts tested, significantly increased the accumulation of [3H]paclitaxel, a P-gp substrate, in the cells. Among nine extracts, Z01 and Z02, extracts from Curcuma comosa and Kaempferia marginata (Zingiberaceae family), respectively, potently increased the accumulation. In addition, Z01 and Z02 increased the accumulation of other P-gp substrates, rhodamine 123 and doxorubicin, in PR-HepG2 cells in a concentration-dependent manner. Increased accumulation of rhodamine 123 and doxorubicin by Z01 and Z02 was also confirmed by confocal laser scanning microscopy. The effect of Z01 and Z02 pretreatment on the expression of MDR1 mRNA was also examined. The expression of MDR1 mRNA was not affected by the treatment of PR-HepG2 cells with these extracts for 48 hours. Cytotoxicity of paclitaxel was examined by XTT and protein assays in the absence and presence of Z02. Z02 potentiated the cytotoxicity of paclitaxel in PR-HepG2 cells. These results suggest that Curcuma comosa and Kaempferia marginata belonging to Zingiberaceae are useful sources to search for new P-gp modulator(s) that can be used to overcome multidrug resistance of cancer cells.

Factors affecting the expression and function of P-glycoprotein in rats: drug treatments and diseased states.

The expression and function of P-glycoprotein (P-gp), an ATP-dependent efflux pump, were examined in rats pretreated with dexamethasone (DEX), an inducer of P-gp, and in rats with glycerol-induced acute renal failure (ARF) and with CCl4-induced acute hepatic failure (AHF). DEX pretreatment increased the P-gp level and its functional activity in the intestine. In contrast, in ARF and AHF rats, the in vivo P-gp function was systemically suppressed, even though the level of P-gp remained unchanged or rather increased. In Caco-2 cells, the plasma collected from diseased rats exhibited a greater inhibitory effect on P-gp function than did plasma from control rats. A higher-plasma level of corticosterone, an endogenous P-gp substrate/inhibitor, was observed in the disease rats. These findings indicate that the actual in vivo function of P-gp cannot be predicted merely from the expression level of P-gp, and suggest that some endogenous P-gp-related compounds such as corticosterone participate in the regulation of in vivo P-gp function in diseased states.

Pharmacokinetic interaction of cytochrome P450 3A-related compounds with rhodamine 123, a P-glycoprotein substrate, in rats pretreated with dexamethasone.

The effect of pretreatment with dexamethasone (DEX) on drug-drug interactions between rhodamine 123 (Rho123), a P-glycoprotein (P-gp) substrate, and midazolam, a cytochrome P450 (CYP) 3A substrate, or verapamil, a P-gp/CYP3A substrate, was studied in rats. Rats were pretreated with DEX (100 mg/kg/day, oral) for 2 days. Western blot analysis with a monoclonal antibody for P-gp, C219, revealed that DEX pretreatment increased P-gp level in the intestine 1.9-fold, but not in the liver. In vitro metabolism study of erythromycin in microsomal suspensions indicated the 9.7-fold increase of CYP3A activity in the liver, but not in the intestine, by DEX pretreatment. In an in vivo study, DEX pretreatment increased P-gp-mediated exsorption clearance of Rho123 from blood to the intestinal lumen approximately 2-fold, but not biliary clearances, in good agreement with the results of Western blot analysis. In untreated rats, midazolam (100 microM) or verapamil (30 or 100 microM) added in the intestinal perfusate (single perfusion) decreased the exsorption clearance and biliary clearance of Rho123 by approximately 30 to 50%. In DEX-pretreated rats, however, the inhibitory potency of midazolam in the liver significantly decreased compared with that in untreated rats, although the potency in the intestine did not change. The inhibitory potency of verapamil decreased both in the intestine and liver by DEX pretreatment. In conclusion, it was demonstrated that DEX pretreatment affects not only P-gp-mediated disposition of Rho123 but also pharmacokinetic interactions of P-gp/CYP3A-related compounds with Rho123, probably because concentrations of substrates/inhibitors at target sites such as the intestine and liver are varied.

Expression and function of P-glycoprotein in rats with glycerol-induced acute renal failure.

The effect of glycerol-induced acute renal failure on P-glycoprotein expression and function was evaluated in rats. The in vivo function of P-glycoprotein was evaluated by measuring renal secretory and biliary clearance and brain distribution of rhodamine 123 (Rho-123), a P-glycoprotein substrate, under a steady-state plasma concentration. In acute renal failure rats, the P-glycoprotein level increased 2.5-fold in the kidney, but not in the liver and brain. In contrast, P-glycoprotein function in these tissues was suppressed. Interestingly, not only the renal but also the biliary clearance of Rho-123 was correlated with the glomerular filtration rate. In Caco-2 cells, plasma from renal failure rats exhibited a greater inhibitory effect on P-glycoprotein-mediated transport of Rho-123 than did plasma from control rats. In conclusion, P-glycoprotein function was systemically suppressed in acute renal failure, even though the level of P-glycoprotein remained unchanged or rather increased. This may be due to the accumulation of some endogenous P-glycoprotein substrates/modulators in the plasma in disease states.

Enhancing effect of 5 alpha-cyprinol sulfate on mucosal membrane permeability to sodium ampicillin in rats.

Effect of 5 alpha-cyprinol sulfate, a bile alcohol sulfate specific to carp bile, on rectal membrane permeability to sodium ampicillin (AMP Na) was examined in rats. AMP Na is not easily absorbed through rat rectal membrane without aid. 5 alpha-Cyprinol sulfate significantly enhanced the rectal membrane permeability to AMP Na even at a low concentration (6.25 mM), though sodium taurocholate needed a higher concentration (25 mM). Co-administration of phosphatidylcholine significantly suppressed the enhancing action of both sodium taurocholate and 5 alpha-cyprinol sulfate. On the other hand, calcium ion did not suppress the action of 5 alpha-cyprinol sulfate, although it did clearly suppress the action of sodium taurocholate. In conclusion, 5 alpha-cyprinol sulfate was found to have a potent enhancing effect on mucosal membrane permeability to water-soluble compounds. The enhancing mechanism of 5 alpha-cyprinol sulfate appeared to be different from that of sodium taurocholate.

Iontophoretic transdermal delivery of salicylic acid dissolved in ethanol-water mixture in rats.

The usefulness of iontophoresis is restricted to highly water-soluble compounds, since drugs are generally applied as an aqueous solution in a drug electrode. In the present study, salicylic acid (SA) dissolved in ethanol-water mixture was loaded in a drug electrode, and the effect of ethanol on the iontophoretic transdermal delivery of SA was evaluated. Ethanol at a concentration of 10 or 30% showed no significant effect on the iontophoretic transdermal delivery of SA compared to that in the absence of ethanol, but 40 or 70% ethanol increased it significantly. The current density passing through in vivo during iontophoretic treatment decreased with increase in ethanol concentrations. These results suggested that the enhanced transdermal absorption of SA iontophoretically by the presence of ethanol in a drug solution is not due to the increased current density in vivo, but probably due to the direct action of ethanol on the stratum corneum. In conclusion, addition of ethanol to a drug solution at an appropriate concentration was proved to enhance the iontophoretic transdermal delivery of SA. A mixture of ethanol and water can dissolve many poorly water-soluble drugs, and therefore it would be able to expand the application of iontophoresis to include many drugs that are poorly soluble in water.

Transport of rhodamine 123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers in the presence of cytochrome P-450 3A-related compounds.

Effects of cytochrome P-450 3A- and P-glycoprotein (P-gp)-related compounds, erythromycin, midazolam, ketoconazole, verapamil, and quinidine, on transport of rhodamine 123 (Rho-123), a P-gp substrate, were studied in rat intestine and in Caco-2 cells. Ileum was mainly used in rat studies because this segment showed greater P-gp-mediated Rho-123 transport. In an in vitro everted rat ileum, all the compounds examined significantly inhibited the transport of Rho-123 from serosal to mucosal surfaces across the intestine, with different inhibitory potencies among these compounds. In an in vivo rat study, the exsorption of Rho-123 from blood to the intestinal lumen, which was evaluated as exsorption clearance of Rho-123 under a steady-state plasma concentration of Rho-123, was also inhibited when these compounds were added to the intestinal lumen. Similarly, transepithelial transport of Rho-123 from the basolateral to apical side across Caco-2 cell monolayers was inhibited by these compounds. A linear relationship was observed in their inhibitory potencies on Rho-123 transport between in vitro and in vivo studies using rat ileum and between studies with rat ileum and Caco-2 cells. P-gp-mediated transport across the intestine was found to be inhibited not only by P-gp-related but also by all the cytochrome P-450 3A-related compounds examined. Within experimental error, the relative inhibitory potencies were the same between the studies with rat ileum (in vivo, in vitro) and those with Caco-2 cells. Thus, it is suggested that the function of P-gp and its sensitivity to these drugs may be similar in rat intestine and Caco-2 cells.

Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells.

The effect of cytochrome P-450 3A (CYP3A) substrates (erythromycin, midazolam) and an inhibitor (ketoconazole) on P-glycoprotein-mediated transport was studied in Caco-2, the human colon adenocarcinoma cell line expressing various functions of differentiated intestinal epithelial cells. The involvement of P-glycoprotein in the transport of these drugs was also examined. The basal-to-apical transport of rhodamine 123, a P-glycoprotein substrate, was inhibited by erythromycin, midazolam and ketoconazole, as well as by P-glycoprotein inhibitors such as verapamil. The apical-to-basal transport of rhodamine 123 was increased by these drugs. The transepithelial transport of erythromycin and midazolam, but not of ketoconazole, was much greater from the basal to apical side than from the apical to basal side. The inhibitory effect of verapamil was observed on the basal to apical transport of erythromycin, but not on midazolam and ketoconazole transport. In conclusion, erythromycin, midazolam and ketoconazole could interact with P-glycoprotein-mediated transport, and P-glycoprotein could be, at least in part, involved in the transport of erythromycin, but not of midazolam and ketoconazole, in the intestinal epithelia.

N-acetyl-L-gamma-glutamyl derivatives of p-nitroaniline, sulphamethoxazole and sulphamethizole for kidney-specific drug delivery in rats.

Kidney-specific delivery of p-nitroaniline, sulphamethoxazole and sulphamethizole after either intravenous administration of the L-gamma-glutamyl or N-acetyl-L-gamma-glutamyl derivatives or the parent drugs has been examined in a rat model. All L-gamma-glutamyl derivatives were converted to the corresponding parent drugs within 60 min whereas the N-acetyl-L-gamma-glutamyl derivatives were fairly stable in the systemic circulation after parenteral administration. Concentrations of p-nitroaniline and sulphamethoxazole 20 min after administration of the parent drugs were somewhat higher in the kidney than in the liver and lung. The concentration of sulphamethizole in the kidney was dramatically higher than those in the hepatic and pulmonary tissue. Kidney-specific delivery of the drugs of interest was evaluated by determining the tissue concentrations of the released parent drug and the total drug levels (i.e. drug levels after hydrolysis of all conjugate to the parent drug). For L-gamma-glutamyl-p-nitroaniline released renal levels of p-nitroaniline and total p-nitroaniline concentrations were both higher than those obtained after p-nitroaniline dosing. Use of L-gamma-glutamylsulphamethoxazole resulted in higher total sulphamethoxazole concentrations in the kidney, but did not lead to an increase in released (unconjugated) sulphamethoxazole levels. In contrast, no kidney-selective distribution was observed for L-gamma-glutamylsulphamethizole. Markedly increased kidney distribution was observed for both N-acetyl-L-gamma-glutamyl-p-nitroaniline and N-acetyl-L-gamma-glutamylsulphamethoxazole and the liver and lung concentrations were correspondingly reduced in comparison with parent drug dosing. Use of the N-acetyl-L-gamma-glutamyl-p-nitroaniline conjugate increased the concentration of p-nitroaniline in the kidney to the same extent as did L-gamma-glutamyl-p-nitroaniline. In conclusion, N-acetyl-L-gamma-glutamyl derivatization of certain compounds seems to be useful for kidney-specific drug delivery and preliminary data suggests that lipophilic drugs are better substrates than hydrophilic compounds. Results related to the selectivity of tissue distribution of the derivatives and species differences are discussed.

Topical delivery of keloid therapeutic drug, tranilast, by combined use of oleic acid and propylene glycol as a penetration enhancer: evaluation by skin microdialysis in rats.

Topical delivery of tranilast (N-(3,4-dimethoxycinnamoyl)anthranic acid), an inhibitor of collagen synthesis and a therapeutic drug for keloid and hypertrophic scar, was examined, in rats, with oleic acid alone or a combination of oleic acid and propylene glycol as penetration enhancer. Evaluation was by measurement of the concentration of tranilast in plasma and in the dialysate from skin microdialysis. When tranilast at a dose of 1.5 mg was applied topically as an ethanol solution containing 5% polyvinylpyrrolidone on a dorsal skin surface (2.25 cm2), the maximum concentration of tranilast in skin dialysate was approximately 2 microM. When 10 or 20% oleic acid was added to the same ethanol solution the maximum concentration of tranilast in the dialysate increased to 10-20 microM, and this value was further increased to 60 microM by the addition of a combination of oleic acid (10 or 20%) and propylene glycol (10%) to the solution. With the combination of oleic acid and propylene glycol the area under the plot of the concentration of tranilast in skin dialysate against time between 0 and 4 h (AUC0-4) was more than 400-fold that after intravenous administration. The transdermal bioavailability of tranilast as assessed by the AUC0-4 of tranilast in plasma, was 0.2% of the dose applied in the ethanol solution, 3-5% of that applied in the ethanol solution containing oleic acid, and 14-16% of that applied in the ethanol solution containing both oleic acid and propylene glycol. These results suggest that the topical delivery of tranilast with an absorption enhancer such as a mixture of oleic acid and propylene glycol might be a more effective medication than oral administration of tranilast for the treatment of keloid and hypertrophic scar.

Effect of ointment bases on topical and transdermal delivery of salicylic acid in rats: evaluation by skin microdialysis.

Microdialysis has been used to determine the concentration of salicylic acid in skin tissue and plasma periodically for 4 h to evaluate the effect of ointment bases on topical and transdermal delivery of salicylic acid. The ointment bases examined were solbase (water-soluble), poloid and white petrolatum (oleaginous), hydrophilic poloid (water in oil (w/o) type emulsion lacking water) and absorptive ointment (w/o-type emulsion containing water). The ointments (0.1 g) containing 25 micromol salicylic acid were applied for 2 h to the surface of rat skin (1 cm2) with (intact) or without the stratum corneum. For intact skin, the extent of topical delivery from different ointments, evaluated by the area under the concentration-time curve (AUC) of salicylic acid in the skin tissue (AUCskin), increased in the order solbase << white petrolatum, poloid, hydrophilic poloid << absorptive ointment. The ratio of AUCskin (topical delivery) to the AUC of salicylic acid in plasma (AUCplasma, transdermal delivery) varied remarkably among the different bases, the greatest ratio being observed for absorptive ointment. When the ointments were applied to skin surface without stratum corneum, AUCskin for solbase was much higher (about 45 times that for intact skin), whereas only a small (two-fold) increase was observed for poloid and hydrophilic poloid and the increase was negligible for white petrolatum and absorptive ointment. For skin without the stratum corneum, the ratio AUCskin/AUCplasma for the different ointments was comparable, although the magnitudes of AUCskin and AUCplasma still varied substantially. The variance of AUC values arises as a result of the different rates of release of salicylic acid from the bases. These results indicate that: the topical and transdermal delivery of salicylic acid in intact skin varies substantially among different ointment bases, and the greatest topical delivery is observed for absorptive ointment; use of absorptive ointment increases the retention of salicylic acid in the stratum corneum; and the stratum corneum functions strongly as a penetration barrier for solbase, moderately for poloid and hydrophilic poloid, and less for absorptive ointment and white petrolatum.

Decanoic acid induced enhancement of rectal absorption of hydrophilic compounds in rats.

The enhancing effect of decanoic acid (C10) on the rectal absorption of phenolsulfonphtalein (PSP) was analyzed with a pharmacokinetic model. The transfer index of PSP from the rectal lumen to the epithelial layer in the presence of C10 was proportional to the product of the C10 disappearance rate from the rectal lumen and its calcium ion sequestration capacity. The enhancement of rectal PSP absorption by different doses of C10 was also predictable by using a permeability index, Pa, of PSP, which was defined as a proportionality constant relating transfer index and the product value described above. The Pa values of various hydrophilic compounds with different molecular weights were also estimated from their rectal bioavailability in the presence of C10. A linear relationship was observed between Pa values and reciprocal values of the square root of individual molecular weight. These findings suggest that the Pa index is related to the permeant's diffusion coefficient through paracellular aqueous openings formed by C10.

Pharmacokinetic analysis of the absorption enhancing action of decanoic acid and its derivatives in rats.

The enhancing action of decanoic acid (C10) and its derivatives on mucosal absorption of phenolsulfonphthalein (PSP) in the jejunum or colon was analyzed using pharmacokinetics in rats. After administration of a solution containing PSP and an enhancer [C10, 2-hydroxydecanoic acid (2-OHC10), or 3-hydroxydecanoic acid (3-OHC10)] into the jejunal or colonic loop, the amounts of PSP and enhancer remaining in the loop and/or plasma PSP concentration were determined periodically. 2-OHC10 exhibited a greater absorption enhancing potency than C10, while 3-OHC10 was less effective. Disappearance of residual PSP from the loop ceased after complete absorption of the enhancer. The enhancer-induced disappearance rate constant of PSP correlated well with the product of the enhancer disappearance rate and its capacity to sequester calcium ions. In conclusion, the enhancement of PSP mucosal absorption by C10 and its derivatives is consistent with a pharmacokinetic model, assuming that the enhanced membrane permeability of PSP depends on the enhancer disappearance kinetics from the loop and its calcium ion sequestration capacity.