Hisanao Kishimoto

Effects of nitric oxide on mucosal barrier dysfunction during early phase of intestinal ischemia/reperfusion

Ischemia/reperfusion (I/R) injury must be overcome for successful small intestinal transplantation. During intestinal I/R, the expression level of nitric oxide (NO) is increased, and vermiculation of the mucosal tract is induced by NO. Although NO has many beneficial effects on intestinal I/R injury, its role in intestinal I/R injury is controversial. Therefore, in the present study, we examined changes in the tight junctions (TJ) and P-glycoprotein (P-gp) by aminoguanidine (AG), which can be considered a selective inducible NO synthase inhibitor during intestinal I/R, to clarify the effect of NO on mucosal barrier dysfunction during intestinal I/R. A mucosal lesion was induced by intestinal I/R. The protein expression levels of the claudin family organizing TJ and P-gp, were decreased, and their functions were also decreased. Through the inhibition of NO generation by AG in the above mucosal lesion, TJ and P-gp dysfunction was significantly inhibited. NO participated in opening TJ and decreasing P-gp function and expression induced during intestinal I/R. Therefore, it is important to consider the level of NO generation in the ileal mucosa in drug therapy for intestinal I/R injury.

Changes in protein and mRNA expression levels of claudin family after mucosal lesion by intestinal ischemia/reperfusion

Ischemia/reperfusion (I/R) injury of the intestine is the leading cause of organ dysfunction after restoration of blood flow after diverse events, including shock and intestinal transplantation. I/R injury must be overcome for successful small intestinal transplantation. Tight junctions (TJ) are the most apical component of the intercellular junctional complex in epithelial cells; they establish cell polarity and functioning as major determinants of epithelial barrier function. Among the proteins that comprise TJ, the claudin family is thought to play a crucial role in homeostasis in multicellular organisms. Therefore, the aim of this study was to examine the changes in function of TJ and behavior of the claudin family during intestinal I/R. Wistar/ST rats underwent intestinal ischemia by using the spring scale and surgical suture for 1h, followed by 24h of reperfusion. We examined the changes in area under the blood concentration curve (AUC) after oral administration of FD-4, which is a paracellular marker, and claudin-1, -2, -4, and -7 mRNA and protein expression levels in ileum. The structure of ileal mucosa was partly damaged and its function was diminished by intestinal I/R until 3h after reperfusion, but were almost recovered 24h after reperfusion. However, a time difference was shown between the recoveries of mucosal structure and function. Furthermore, a difference in the expression among various kinds of claudin was found. It was suggested that claudin-4 and multi-PDZ domain protein, which is a scaffolding protein, regulate intestinal paracellular permeability during intestinal I/R. Moreover, the changes in the expression level of claudin-2 were unique.

Effect of intestinal ischaemia/reperfusion on P-glycoprotein-mediated ileal excretion of rhodamine 123 in the rat

Objectives We have shown that ischaemia/reperfusion in the small intestine at an early phase, such as 1 h after reperfusion, induced not only functional changes in the membrane, such as P‐glycoprotein (P‐gp) dysfunction, but also decreased expression of P‐gp protein and mdr1a mRNA. In the present study we examined whether intestinal ischaemia/reperfusion modifies the P‐gp‐mediated ileal excretion transport system in rats beyond 1 h after reperfusion.

Effects of pharmaceutical excipients on membrane permeability in rat small intestine.

Pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes to the tight junction (TJ) and P-glycoprotein (P-gp), which can affect drug disposition. In this study, we examined the effects of 20 common pharmaceutical excipients from different classes on mucosal membrane and the differences of such effects among regions of the small intestine. We used the in vitro sac method in rat jejunum and ileum to study the effects of excipients on the membrane permeation of 5(6)-carboxyfluorescein (5-CF). 5-CF was used as a model of water-soluble compounds. In some dosage conditions of methyl-β-cyclodextrin, the membrane permeability of 5-CF was significantly increased in the jejunum, but such change was not observed in the ileum. Similarly, in the cases of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and croscarmellose sodium, the membrane permeability of 5-CF was significantly increased in the jejunum, but no change was observed in the ileum. On the other hand, in both the jejunum and the ileum, the membrane permeation of 5-CF was decreased with 0.02% (w/v) hydroxypropyl cellulose, but significantly increased with it at 0.20% (w/v). It was shown that excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and these effects on absorption differed among regions of the small intestine. Moreover, in the case of 20 excipients, not only an increase in membrane permeability but also a decrease was observed. Therefore, it was suggested that a more effective formulation could be designed by changing the combination of excipients.

Changes in the expression levels of tight junction components during reconstruction of tight junction from mucosal lesion by intestinal ischemia/reperfusion.

Tight junction (TJ) is composed of the most apical components of the intercellular junctional complex in epithelial cells; TJ has cell polarity and functions as a major determinant of epithelial barrier function. In this study, to clarify the components of TJ required for its reconstruction and functional acquisition, we examined the changes in intestinal mucosal structure that depended on mucosal lesion by intestinal I/R, that is, the changes in mRNA and protein expression of the claudin family and scaffold proteins. We used an in vivo intestinal I/R model made using the spring scale and surgical sutures, and examined the mRNA and protein expression levels of TJ components by real-time RT-PCR and Western blotting, respectively. Changes in mRNA and protein expression levels of TJ components by intestinal I/R were observed. Among them, characteristic changes were observed in claudin-2 and claudin-4. In addition, the expression behavior of multi-PDZ domain protein (MPDP) mRNA was similar to that of claudin-4. In conclusion, in the recovery process of TJ from mucosal lesion by intestinal I/R, it was suggested that claudin-2 and claudin-4 strongly participate in the reconstruction and functional acquisition of TJ, respectively. Furthermore, it was suggested that MPDZ, which is scaffold protein, also has an important role in these processes.

Characteristics of reversible absorption-enhancing effect of sodium nitroprusside in rat small intestine.

Nitric oxide (NO) donors increase the permeability of water-soluble compounds with neither loss of cell viability nor lactate dehydrogenase release. In addition, the rectal absorption of insulin has been reported to be remarkably enhanced in the presence of NO donors such as 1-Hydroxy-3-(3-aminopropyl)-3-isopropyltriazene 2-oxide (NOC5) and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine (NOC12). In this study, we examined the effect of sodium nitroprusside (SNP), which is used in clinical situations as a vasodilator, as a model NO donor on the ileal mucosa of rats. We used an in situ closed loop method in rat ileum to study changes in the permeability of fluorescein isothiocyanate dextran 4000 (FD-4) as a paracellular marker. The effect of SNP (1 and 10mg/kg) on the protein expression level of the claudin family was examined by Western blotting. The membrane permeation of FD-4 was increased but no mucosal lesion was observed upon the administration of SNP. Moreover, the protein expression level of the claudin family was not changed by the administration of SNP. When SNP was removed 2h after its administration, no significant change in the membrane permeation of FD-4 was observed. Moreover, no decrease of ileal membrane resistance or disruption of membrane structure was observed. The absorption-enhancing effect of SNP was associated with low injury and low toxicity. The reversibility of the effect of SNP was observed. Consequently, it was shown that SNP can be a useful absorption enhancer.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin–luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23 μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.

Absorption-Enhancing Effect of Nitric Oxide on the Absorption of Hydrophobic Drugs in Rat Duodenum.

Nitric oxide (NO), an endogenous gas that plays a versatile role in the physiological system, has the ability to increase the intestinal absorption of water-soluble compounds through the paracellular route. However, it remains unclear whether NO can enhance the absorption of hydrophobic drugs through the transcellular route. In this study, we examined the absorption-enhancing effect of NO on intestinal permeability of hydrophobic drugs in rat intestine. The pretreatment of rat gastrointestinal sacs with NOC7, a NO-releasing reagent, significantly increased the permeation of griseofulvin from mucosa to serosa in the sacs prepared from the duodenum, but not in those prepared from the other regions such as jejunum, ileum, and colon. The absorption-enhancing effect of NOC7 on the duodenal permeation varied depending on the hydrophobicity of the drugs used. Furthermore, NOC7 treatment was found to be apparently ineffective on the griseofulvin permeation in the duodenum pretreated with dithiothreitol (DTT) that was used as a mucus remover, even though the permeation was increased by pretreatment with DTT alone. These results suggest that NO increases the absorption of hydrophobic drugs through the transcellular route in the duodenum by modulating the mucus layer function.

Changes in absorption and excretion of rhodamine 123 by sodium nitroprusside.

Nitric oxide (NO) donors increase the permeability of water-soluble compounds with neither loss of cell viability nor lactate dehydrogenase release, but the involved mechanism is not fully understood. In this study, we focused on permeation via the transcellular route and P-glycoprotein, which is a typical ABC transporter. We examined the effect of sodium nitroprusside (SNP), which is an NO donor, on the membrane permeation of rhodamine 123 (Rho123), a representative P-gp substrate, and the change in expression level of ileal P-gp. We used an in situ closed loop method in rat ileum to study changes in the permeation of Rho123. The effects of SNP (1 and 10mg/kg) on the mdr-1a mRNA and P-gp protein expression levels were examined by real-time RT-PCR and Western blotting, respectively. The absorption and excretion of Rho123 were significantly increased in an SNP dose-dependent manner when compared with those with no addition, but no changes in protein expression level of P-gp in ileal BBM were observed by SNP administration. The relative activity of P-gp was not changed by SNP administration. On the other hand, the expression level of mdr-1a mRNA was induced by SNP administration. We indicated that SNP could increase the mucosal permeation of Rho123 via the transcellular route without an influence on P-gp, and we showed that this effect is temporary. SNP has no influence on P-gp function and protein expression level in the short term, but they may change in the long term.