In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives.

Hepatocyte uptake of phalloidin is carried out mainly by OATP1B1. We have used this compound as a prototypic substrate and assayed the ability to inhibit OATP-mediated phalloidin transport of four bile acid derivatives (BALU-1, BALU-2, BALU-3 and BALU-4) that showed positive results in preliminary screening. Using Xenopus laevis oocytes for heterologous expression of transporters, BALUs were found to inhibit taurocholic acid (TCA) transport by OATP1B1 (but not OATP1B3) as well as by rat Oatp1a1, Oatp1a4 and Oatp1b2. The study of their ability to inhibit sodium-dependent bile acid transporters revealed that the four BALUs induced an inhibition of rat Asbt-mediated TCA transport, which was similar to TCA-induced self-inhibition. Regarding human NTCP and rat Ntcp, BALU-1 differs from the other three BALUS in its lack of effect on TCA transport by these proteins. Using HPLC-MS/MS and CHO cells stably expressing OATP1B1 the ability of BALU-1 to inhibit the uptake of phalloidin itself by this transporter was confirmed. Kinetic analysis using X. laevis oocytes revealed that BALU-1-induced inhibition of OATP1B1 was mainly due to a competitive mechanism (Ki=8 microM). In conclusion, BALU-1 may be useful as a pharmacological tool to inhibit the uptake of compounds mainly taken up by OATP1B1 presumably without impairing bile acid uptake by the major carrier accounting for this process, i.e., NTCP.

Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity.

Phalloidin causes severe liver damage characterized by marked cholestasis, which is due in part to irreversible polymerization of actin filaments. Liver uptake of this toxin through the transporter OATP1B1 is inhibited by the bile acid derivative BALU-1, which does not inhibit the sodium-dependent bile acid transporter NTCP. The aim of the present study was to investigate whether BALU-1 prevents liver uptake of phalloidin without impairing endogenous bile acid handling and hence may have protective effects against the hepatotoxicity induced by this toxin. In anaesthetized rats, i.v. administration of BALU-1 increased bile flow more than taurocholic acid (TCA). Phalloidin administration decreased basal (-60%) and TCA-stimulated bile flow (-55%) without impairing bile acid output. Phalloidin-induced cholestasis was accompanied by liver necrosis, nephrotoxicity and haematuria. In BALU-1-treated animals, phalloidin-induced cholestasis was partially prevented. Moreover haematuria was not observed, which was consistent with histological evidences of BALU-1-prevented injury of liver and kidney tissue. HPLC-MS/MS analysis revealed that BALU-1 was secreted in bile mainly in non-conjugated form, although a small proportion (<5%) of tauro-BALU-1 was detected. BALU-1 did not inhibit the biliary secretion of endogenous bile acids. When highly choleretic bile acids, - ursodeoxycholic (UDCA) and dehydrocholic acid (DHCA) - were administered, they were found less efficient than BALU-1 in preventing phalloidin-induced cholestasis. Biliary phalloidin elimination was low but it was increased by BALU-1>TCA>DHCA>UDCA. In conclusion, BALU-1 is able to protect against phalloidin-induced hepatotoxicity, probably due to an inhibition of the liver uptake and an enhanced biliary secretion of this toxin.

The suprabasal layer of corneal epithelial cells represents the major barrier site to the passive movement of small molecules and trafficking leukocytes.

AIMS: To investigate the site of barrier function to the passive diffusion of a small molecule (phalloidin) in the corneal epithelium in the mouse. METHODS: Penetration of phalloidin (molecular weight 1115 daltons) into the cornea was evaluated by studying fluorescent binding of phalloidin to actin in tissue sections, in whole mount preparations, and in the fixed intact globe by confocal microscopy. In addition, the location of tight junction proteins in the individual layers of the corneal epithelium was determined by immunohistochemistry. RESULTS: Phalloidin staining of corneal sections was positive in all corneal layers in tissue sections and in all layers of the corneal epithelium except the suprabasal layer in excised fixed whole mounts of the cornea. However, when phalloidin staining was attempted in intact fixed globes, before excision of the cornea for whole mount preparation, only the most superficial layer of cells was stained indicating that phalloidin could not penetrate the tissue beyond the suprabasal epithelial layer. Detergent (Triton X-100) treatment of the excised cornea and the intact fixed globe, allowed penetration of phalloidin into the suprabasal epithelial layer. Tight junction proteins occludin, ZO-1 and claudin were present in most layers of the cornea but while ZO-1 and occludin were distributed in a typical pericellular pattern, claudin seemed to be particularly prominent in the suprabasal layer and appeared only as a discontinuous punctate pericellular pattern in the superficial layer. Intraepithelial leukocytes were detected in the superficial epithelium and the basal epithelium but not in the suprabasal epithelium. CONCLUSION: The suprabasal epithelium cell layer appears to represent the main barrier site to the passage of small molecules and cells in the mouse cornea and this property may be attributable to prominent claudin expression in this layer.

Identification of phalloidin uptake systems of rat and human liver.

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na+/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [3H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [3H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [3H]demethylphalloin concentrations was saturable with apparent Km values of 5.7 microM (Oatp1b2), 17 microM (OATP1B1) and 7.5 microM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [3H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.

Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability.

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the outer retina in the mouse.

Mice represent a valuable species for studies of development and disease. With the availability of transgenic models for retinal degeneration in this species, information regarding development and structure of mouse retina has become increasingly important. Of special interest is the differentiation and synaptogenesis of photoreceptors since these cells are predominantly involved in hereditary retinal degenerations. Thus, some of the keys to future clinical management of these retinal diseases may lie in understanding the molecular mechanisms of outer retinal development. In this study, we describe the expression of markers for photoreceptors (recoverin), horizontal cells (calbindin), bipolar cells (protein kinase C; PKC) and cytoskeletal elements pivotal to axonogenesis (beta-tubulin and actin) during perinatal development of mouse retina. Immunocytochemical localization of recoverin, calbindin, PKC and beta-tubulin was monitored in developing mouse retina (embryonic day (E) 18.5 to postnatal day (PN) 14), whereas f-actin was localized by Phalloidin binding. Recoverin immunoreactive cells, presumably the photoreceptors, were observed embryonically (E 18.5) and their number increased until PN 14. Neurite projections from the immunoreactive cells towards the outer plexiform layer (OPL) were noted at PN 0 and these processes reached the OPL at PN 7 coincident with histological evidence for the differentiation of the OPL. Outer segments, all the cell bodies in the ONL, as well as the OPL were immunoreactive to recoverin at PN 14. Calbindin immunoreactive horizontal cells were also present in E 18.5 retinas. These cells became progressively displaced proximally as the ONL developed. A calbindin immunoreactive plexus was seen in the OPL at PN 7. PKC immunoreactive bipolar cells developed postnatally, becoming distinguished at PN 7. Both beta-tubulin and actin immunoreactive cells were present in the IPL as early as E 18.5; however, appearance of processes labeled with these markers in the OPL was delayed until PN 7, concurrent with the first appearance of photoreceptor neurites, development of the horizontal cell plexus, and development of synaptophysin immunoreactivity at this location. These results provide a developmental timeframe for the expression of recoverin, calbindin, synaptophysin, beta-tubulin and actin. Our findings suggest that the time between PN 3 and PN 7 represents a critical period during which elements of the OPL are assembled.

The 2-phenylbenzotriazole-type water pollutant PBTA-2 has cytochalasin B-mimetic activity.

The 2-phenylbenzotriazole (PBTA)-type water pollutant, 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), has been recently identified in samples from the Nishitakase River in Kyoto, Japan, and shows potent mutagenic activities in Salmonella typhimurium in the presence of a microsomal metabolizing system (S9 mix). In the present study, we conducted the in vitro micronucleus (MN) test on PBTA-2 in the absence and presence of S9 mix in two Chinese hamster cell lines, CHL and V79-MZ. In the MN test, PBTA-2 was weakly positive in CHL cells and strongly positive in V79-MZ cells. Because the positive results were accompanied by a statistically significant increase in the number of polynuclear (PN) and/or mitotic (M) cells, we examined treated cells in metaphase to see if numerical chromosome aberrations were being induced. We found that PBTA-2 induces polyploidy in both CHL and V79-MZ cells. A detailed analysis of MN preparations showed that in CHL cells, PBTA-2 predominantly induces equal-sized binucleated cells. Rhodamine phalloidin staining revealed that PBTA-2 causes actin filament abnormalities in both cell lines similar to those caused by cytochalasin B. Cytochalasin B induced PN cells predominantly and dose dependently, and almost all the cells were equal-sized and binucleate. The results suggest that PBTA-2 has cytochalasin B-mimetic activity, although agents affecting actin filaments, such as cytochalasins, phallotoxins and chloropeptide, have been derived only from molds so far. This study also suggests that our MN test protocol may be used to identify chemicals that have cytochalasin B-mimetic activity as well as those that induce numerical aberrations.

Permeation of fluorophore-conjugated phalloidin into live hair cells of the inner ear is modulated by P2Y receptors.

Phalloidin, a toxin isolated from the death cap mushroom, Amanita phalloides, binds to filamentous actin with high affinity, and this has made fluorophore-conjugated phalloidin a useful tool in cellular imaging. Hepatocytes take up phalloidin via the liver-specific organic anion transporting polypeptide 1b2, but phalloidin does not permeate most living cells. Rapid entry of styryl dyes into live hair cells has been used to evaluate function, but the usefulness of those fluorescence dyes is limited by broad and fixed absorption spectra. Since phalloidin can be conjugated to fluorophores with various spectra, we investigated whether it would permeate living hair cells. When we incubated mouse utricles in 66 nM phalloidin-CF488A and followed that by washes in phalloidin-free medium, we observed that it entered a subset of hair cells and labeled entire hair bundles fluorescently after 20 min. Incubations of 90 min labeled nearly all the hair bundles. When phalloidin-treated utricles were cultured for 24 h after washout, the label disappeared from the hair cells and progressively but heterogeneously labeled filamentous actin in the supporting cells. We investigated how phalloidin may enter hair cells and found that P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid and suramin, blocked phalloidin entry, while the P2Y receptor ligands, uridine-5'-diphosphate and uridine-5'-triphosphaste, stimulated uptake. Consistent with that, the P2Y6 receptor antagonist, MRS 2578, decreased phalloidin uptake. The results show that phalloidin permeates live hair cells through a pathway that requires metabotropic P2Y receptor signaling and suggest that phalloidin can be transferred from hair cells to supporting cells in culture.

Characterization of organic anion transporting polypeptide 1b2-null mice: essential role in hepatic uptake/toxicity of phalloidin and microcystin-LR.

The liver-specific importer organic anion transporting polypeptide 1b2 (Oatp1b2, Slco1b2, also known as Oatp4 and Lst-1) and its human orthologs OATP1B1/1B3 transport a large variety of chemicals. Oatp1b2-null mice were engineered by homologous recombination and their phenotype was characterized. Oatp1b2 protein was absent in livers of Oatp1b2-null mice. Oatp1b2-null mice develop normally and breed well. However, adult Oatp1b2-null mice had moderate conjugated hyperbilirubinemia. Compared with wild-types, Oatp1b2-null mice had similar hepatic messenger RNA expression of most transporters examined except a higher Oatp1a4 but lower organic anion transporter 2. Intra-arterial injection of the mushroom toxin phalloidin (an Oatp1b2-specific substrate identified in vitro) caused cholestasis in wild-type mice but not in Oatp1b2-null mice. Hepatic uptake of fluorescence-labeled phalloidin was absent in Oatp1b2-null mice. Three hours after administration of microcystin-LR (a blue-green algae toxin), the binding of microcystin-LR to hepatic protein phosphatase 1/2a was much lower in Oatp1b2-null mice compared with wild-type mice. In contrast, Oatp1b2-null mice were transiently protected from decrease in bile flow induced by estradiol-17beta-D-glucuronide, a common substrate for Oatps. Oatp1b2-null mice were completely resistant to the hepatotoxicity induced by phalloidin and microcystin-LR, but were similarly sensitive to alpha-amanitin-induced hepatotoxicity compared with wild-type mice. In conclusion, Oatp1b2-null mice display altered basic physiology and markedly decreased hepatic uptake/toxicity of phalloidin and microcystin-LR. Oatp1b2-null mice are useful in elucidating the role of Oatp1b2 and its human orthologs OATP1B1/1B3 in hepatic uptake and systemic disposition of toxic chemicals and therapeutic drugs.

Decreased sensitivity to phalloidin of aged F344/DuCrj rat hepatocytes.

The phalloidin sensitivity of hepatocytes resulting in the formation of cytoplasmic blebs was examined with cells isolated from 73- to 74-week-old and 99- to 100-week-old F344/DuCrj rats of both sexes by a collagenase perfusion method. The cells isolated from aged rats were less sensitive to the toxin than those obtained from 10- to 14-week-old rats. The decrease in the sensitivity was more marked in males than in females, and it appeared at an earlier age in the former than in the latter. Phalloidin consumption experiments showed decreases in the cellular uptake of the toxin in aged rats, and these were more marked in males than in females. The low cellular uptake of the toxin seemed to play an important role in the low sensitivity of the cells in aged rats. Although histological and histochemical examinations showed the development of foci of altered hepatocytes in the aged rat liver, the foci were estimated to account for less than 1.5% of liver tissues. Thus, the decrease in the sensitivity of the cells isolated from whole liver tissues might mainly be attributed to the decrease in the sensitivity of otherwise normal-looking hepatocytes.

Expression and subcellular localization of Ror tyrosine kinase receptors are developmentally regulated in cultured hippocampal neurons.

Ror1 and Ror2 are two novel receptor tyrosine kinases that have been implicated in neuronal differentiation in Caenorhabditis elegans. As a first step toward elucidating their role in the mammalian brain, we analyzed their expression and localization patterns in hippocampal neurons. Our results showed that both receptors are expressed from early stages of development and that their protein levels peak during periods of active synapse formation. Immunocytochemical analysis indicated that Ror1 and Ror2 are highly concentrated in the growth cones of immature neurons and are present throughout the somatodendritic compartment of mature hippocampal cells. Further analysis indicated that they are present not only in the cell membrane but also in Triton- and saponin-insoluble fractions, suggesting that they may be associated with both the cytoskeleton and membrane-bound organelles. Taken collectively, our results suggest that Ror1 and Ror2 might play a role during early stages of development in mammalian central neurons.

Effect of the cytoskeletal fixation agent phalloidin on transcapillary albumin transport and interstitial fluid pressure in anaphylaxis in the wistar rat.

OBJECTIVE: Interstitial fluid pressure (P(if)) plays an important role in controlling interstitial fluid volume. In the early phase of rapid edema formation, such as in dextran-induced anaphylaxis in the Wistar rat, P(if) falls from -0.5 mm Hg to a value between -5 and -10 mm Hg. It is believed that P(if) is controlled by the interaction between connective tissue cells and the extracellular matrix. This hypothesis was tested by studying dextran-induced edema formation and the subsequent changes in P(if) in response to a pretreatment with phalloidin, a reagent that fixes the actin filaments within the cell. METHODS: P(if) was measured in anesthetized female Wistar rats by using a micropuncture technique. The rats were pretreated with phalloidin followed by dextran. Albumin extavasation (E(alb)) was measured as the extravascular space of (125)I-labeled human serum albumin (HSA) after 25 minutes. Total tissue water (TTW) was calculated as the difference between wet weight and dry weight divided by dry weight. Localization of phalloidin was determined histologically by using rhodamine-labeled phalloidin. RESULTS: Control P(if) values of -0.3 +/- 0.6 mm Hg dropped to -3.1 +/- 0.8 mm Hg after dextran treatment (p < 0.05). Pretreatment with phalloidin completely abolished this decrease in P(if), giving values of -0.6 +/- 0.2 mm Hg (p < 0.05, compared to dextran). The E(alb) in control rats of 0.02 +/- 0.02 mL/g DW increased to 1.35 +/- 0.43 mL/g DW after dextran treatment. Pretreatment with phalloidin before dextran treatment lowered the dextran value to 0.59 +/- 0.32 mL/g DW. CONCLUSION: This study shows that pretreatment with phalloidin, before the administration of dextran, abolishes the lowering of P(if) and edema formation, which is detected after i.v. injection of dextran alone.