Carrier-mediated Mechanism Research Articles

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.

Novel insights into intestinal and renal folate transport. Focus on “Apical membrane targeting and trafficking of the human proton-coupled folate transporter in polarized epithelia”

FOLATE IS AN ESSENTIAL MICRONUTRIENT that functions as a coenzyme in DNA and RNA synthesis and in the metabolism of several amino acids, including homocysteine (11). Folate deficiency can occur in humans because of poor dietary intake, malabsorption, metabolic blocks, or increased requirements as seen during pregnancy and lactation (3). Symptoms of deficiency are severe and include megoblastic anemia and neural tube defects in children. Mammals must absorb folate from the diet, because they do not have the capability to synthesize folate. Intestinal absorption occurs by both passive and carriermediated mechanisms, with the second process predominating in the proximal small intestine at normal intake levels (3). Intestinal transport is critical for overall body folate homeostasis, as exemplified by the fact that individuals with hereditary folate malabsorption exhibit signs of folate deficiency (5, 6). Dietary folate exists in the polyglutamate form, which is converted to the monoglutamate form before absorption. Transport across the intestinal epithelium is a two-step process; the putative brush-border membrane transporter is pH dependent (i.e., higher activity at low pH) (12), and movement across the basolateral surface is also a carrier-mediated process that has been previously described (13). Recently, three folate transport systems have been identified in mammals: the reduced folate carrier (RFC) (4, 8), the folate receptor (FR) (7), and the newly described proton-coupled folate transporter (PCFT; also called SLC46A1) (9). The relative role of each of these transport systems in intestinal and renal folate absorption is currently not completely understood (although the FR, which was described in renal cells, has not been identified in the intestine). The recent studies by Subramanian et al. (17) have provided novel evidence that makes a major contribution to our understanding of folate homeostasis. These authors have clearly demonstrated, for the first time, that the human PCFT is a brush-border membrane protein in polarized epithelia; they show that it is functionally expressed on the apical membrane in widely accepted models of the intestinal and renal epithelia. Moreover, in polarized MDCK cells, they noted that the PCFT and the RFC showed differential localization, with the RFC present on the basolateral domain and the PCFT in the apical membrane. Additional experiments were aimed at determining the trafficking motifs of the PCFT and the effect of clinically relevant mutations; results showed that the COOH terminus was not important for apical targeting and that mutation of a region (DXXGR[113]R) associated with hereditary folate malabsorption (the R[113]S mutation has been identified in a human patient) (20) abolished a -turn between membrane-spanning domains 2 and 3 and led to protein retention in the endoplasmic reticulum. Further analyses found that cell surface delivery of the PCFT is dependent on an intact microtubular network. The findings presented in this article reveal new aspects of folate transport that can only be appreciated in the context of previous studies in this field. Prior investigations suggested that the RFC is important in intestinal and renal folate absorption. Some studies found RFC expressed on the intestinal brush-border membrane in mouse (18) and rat (1) intestine, where it is presumably involved in folate absorption, while others described expression at the basolateral surface of renal epithelial cells (2) and at the plasma membrane when expressed in Xenopus oocytes (16). Moreover, the RFC was found to be ontogenically regulated during rat development and was reflective of overall intestinal folate transport (1). Despite these intriguing observations, certain inconsistencies exist between the known properties of intestinal folate transport and those ascribed to the RFC, including the higher activity of the RFC at neutral as opposed to acidic pH (15).

The effect of oxygen annealing on ZnO : Cu and ZnO : (Cu,Al) diluted magnetic semiconductors

Here we report a systematic study of magnetic, electronic and microstructural properties of Cu-doped and Cu and Al codoped ZnO thin films, grown epitaxially on a (0 0 0 1) sapphire substrate by the pulsed laser deposition technique. The films were annealed in oxygen at high temperature (600 °C) and their properties were compared with the as deposited films in order to study the role of defects like oxygen vacancies on the ferromagnetic properties. The doping of ZnO : Cu specimens with Al increased the carrier concentration by three orders of magnitude (from 1017 to 1020 cm−3) without altering the ferromagnetic ordering. On the other hand, a reduction in oxygen vacancies concentration brought about by high temperature annealing in oxygen had a large detrimental effect on ferromagnetism. These results tend to eliminate a free carrier mediated mechanism and point towards a defect mediated mechanism, such as a bound-magnetic-polaron mediated exchange, as being responsible for stabilizing long-range ferromagnetic ordering in these systems.

Phenylphosphonate Transport by Helicobacter pylori

Helicobacter pylori can utilize phenylphosphonate as a sole source of phosphorus, and it is able to transport the phosphonate N-phosphonoacetyl-L-aspartate. However, H. pylori does not have any genes homologous to those of the known pathways for phosphonate degradation in bacteria, indicating that it must have novel pathways for the transport and metabolism of phosphonates. Phenylphosphonate transport by H. pylori was studied in strains LC20, J99 and N6 by the centrifugation through oil method using [(14)C]-labeled phenylphosphonate. The Michaelis constants of transport K(t) and V(max) for phenylphosphonate showed similar kinetics in the three strains. The Arrhenius plot for phenylphosphonate transport rates at permeant concentrations of 50 micromol/L was linear over the temperature range 10-40 degrees C with an activation energy of 3.5 kJ/mol, and a breakpoint between 5 and 10 degrees C. Transport rates increased with monovalent cation size. The effects of various inhibitors were investigated: iodoacetamide, amiloride, valinomycin, and nigericin reduced the rate of phenylphosphonate transport; sodium azide and sodium cyanide increased the transport rate; and monensin had no effect. The kinetics and properties of H. pylori phenylphosphonate transport were characterized, and the data suggested a carrier-mediated transport mechanism.

Mechanism of nicotinic acid transport in human liver cells: experiments with HepG2 cells and primary hepatocytes

This study reports on the functional expression of a specific, high-affinity carrier-mediated mechanism for the transport of niacin (nicotinic acid) in human liver cells. Both human-derived liver HepG2 cells and human primary hepatocytes were used as models in these investigations. The initial rate of transport of nicotinic acid into HepG2 cells was found to be acidic pH, temperature, and energy dependent; it was, however, Na(+) independent in nature. Evidence for the existence of a carrier-mediated system that is specific for [(3)H]nicotinic acid transport was found and included the following: 1) saturability as a function of concentration with an apparent K(m) of 0.73 +/- 0.16 microM and V(max) of 25.02 +/- 1.45 pmol.mg protein(-1).3 min(-1), 2) cis-inhibition by unlabeled nicotinic acid and nicotinamide but not by unrelated organic anions (lactate, acetate, butyrate, succinate, citrate, and valproate), and 3) trans-stimulation of [(3)H]nicotinic acid efflux by unlabeled nicotinic acid. Transport of the vitamin into human primary hepatocytes occurs similarly via an acidic pH-dependent and specific carrier-mediated process. Inhibitors of the Ca(2+)-calmodulin-mediated pathway (but not modulators of the PKC-, PKA-, and protein tyrosine kinase-mediated pathways) inhibited nicotinic acid transport into both HepG2 cells and human primary hepatocytes. Maintenance of HepG2 cells (for 48 h) in growth medium oversupplemented with nicotinic acid (or nicotinamide) did not affect the subsequent transport of [(3)H]nicotinic acid into HepG2 cells. These results show, for the first time, the existence of a specific and regulated membrane carrier-mediated system for nicotinic acid transport in human liver cells.

Evidence for carrier-induced ferromagnetic ordering in Zn1−xMnxO thin films: Anomalous Hall effect

The intrinsic origin of the ferromagnetic ordering in Zn1−xMnxO thin films grown by pulsed-laser deposition was investigated. The ferromagnetic behaviors for a Zn1−xMnxO (x=0.26) film grown at 700 °C under oxygen pressures of 10−1 Torr were observed at 4 and 300 K. The anomalous Hall effect (AHE) was found at temperatures of up to 210 K for the Zn0.74Mn0.26O thin film. The anomalous Hall coefficients (RA) were determined to be approximately proportional to the square of resistivity in the low field region, indicating the side-jump process for the AHE. Our results provide direct experimental evidence that a carrier-mediated mechanism is responsible for the ferromagnetic ordering in Zn1−xMnxO thin films grown by pulsed-laser deposition.

Characterization of a serotonin transporter in the parasitic flatworm, Schistosoma mansoni: Cloning, expression and functional analysis

The biogenic amine serotonin (5-hydroxytryptamine: 5HT) is a widely distributed neuroactive substance of vertebrates and invertebrates. Among parasitic flatworms, in particularly the bloodfluke, Schistosoma mansoni, 5HT is an important modulator of neuromuscular function and metabolism. Previous work has shown that schistosomes take up 5HT from host blood via a carrier mediated mechanism. This transport is thought to contribute to the control of schistosome motility in the bloodstream and is essential for survival of the parasite. Here we provide the first molecular evidence for the existence of a 5HT transporter in S. mansoni. A cDNA showing high homology with plasma membrane serotonin transporters (SERT) from other species was cloned and characterized by heterologous expression in cultured HEK293 cells. Functional studies showed that the recombinant schistosome transporter (SmSERT) mediates specific and saturable [ 3H]-5HT transport with a K t = 1.30 ± 0.05 μM. The heterologously expressed protein was inhibited by classic SERT blockers (clomipramine, fluoxetine, citalopram) and the same drugs also inhibited [ 3H]-5HT uptake by intact schistosomula in culture, suggesting that SmSERT may be responsible for this transport. Conventional (end-point) and real-time quantitative RT-PCR analyses determined that SmSERT is expressed both in the free-living stage (cercaria) and parasitic forms of S. mansoni but the expression level is significantly higher in the parasites. These results suggest that SmSERT is upregulated following cercarial transformation, possibly to mediate the recruitment of exogenous 5HT from the host.

Magnetic, electrical, and microstructural characterization of ZnO thin films codoped with Co and Cu

Here we report on systematic studies of the epitaxial growth and properties of Co and Cu codoped ZnO thin films deposited onto sapphire c-plane single crystals using pulsed-laser deposition. The films display ferromagnetic behavior at room temperature. Detailed atomic scale characterization rules out the presence of clusters and secondary phases as the source of ferromagnetism. Optical measurements and x-ray photoelectron spectroscopy confirm the direct substitution of dopant atoms into Zn lattice sites. At low concentrations of Cu (∼5%) the magnetic moment of Zn1−(0.05+x)Co0.05CuxO materials appears to be additive. At higher concentrations of Cu the net magnetic moment per atom drops off sharply and seems to be relatively insensitive to the Co content. There is a dramatic increase in resistivity of the Co-doped films that accompanies Cu doping. Yet, this change of resistivity does not affect the magnetic moment, suggesting that free carrier mediated mechanism is not a feasible explanation for ferromagnetism in these films. The known presence of high oxygen vacancies in these films does allow for possible defect mediated mechanisms (e.g., bound magnetic polarons) for mediating exchange coupling of the dopant (Co,Cu) ions resulting in room temperature ferromagnetism.

Inward Translocation of the Phospholipid Analogue Miltefosine across Caco‐2 Cell Membranes Exhibits Characteristics of a Carrier‐mediated Process

Miltefosine (hexadecylphosphocholine, HePC) is the first effective oral agent for the treatment of visceral leishmaniasis. The characteristics of HePC incorporation into the human intestinal epithelial cell line Caco-2 were investigated in order to understand its oral absorption mechanism. The results provide evidence for the involvement of a carrier-mediated mechanism, since the association of HePC at the apical pole of Caco-2 cells was (1) saturable as a function of time with a rapid initial incorporation over 5 min followed by a more gradual increase; (2) saturable as a function of concentration over the range studied (2-200 microM) with a saturable component which followed Michaelis-Menten kinetics (apparent K (m) 15.7 micromol/L, V (max) 39.2 nmol/mg protein/h) and a nonspecific diffusion component; (3) partially inhibited by low temperature and ATP depletion, indicating the temperature and energy-dependence of the uptake process. Moreover, we demonstrated, by an albumin back-extraction method, that HePC is internalized via translocation from the outer to the inner leaflet of the plasma membrane and that HePC may preferentially diffuse through intact raft microdomains. In conclusion, our results suggest that incorporation of HePC at the apical membrane of Caco-2 cells may occur through a passive diffusion followed by a translocation in the inner membrane leaflet through an active carrier-mediated mechanism.

Human Small Intestinal Absorption Following Gastric Bypass Surgery

Gastric bypass surgery (GBS) produces a relatively achlorhydric state by formation of a small cardia-fundal pouch with a gastro-jejunal anastomosis. Small intestinal bacterial overgrowth (SIBO) is known to alter vitamin B12 absorption. It has been proposed that gastric acid secretion prevents SIBO. We hypothesized that GBS alters small intestinal micronutrient absorption. This is a retrospective review of patients who underwent Roux-en-Y GBS from 1999-2005. Postoperative patients underwent glucose-hydrogen breath testing to examine upper intestinal (jejunal) bacterial production of methane and hydrogen. Patients’ sex, age, and whole blood thiamine diphosphate levels were obtained 6 months after GBS. Abnormal glucose-hydrogen breath test was defined by fasting breath hydrogen/methane >10 ppm or increased hydrogen/methane of >20 ppm within 60 minutes after 50 grams oral glucose. There were 32 females & 4 males (mean age 47 years; range 22 to 65). All 36 had abnormal glucose-hydrogen breath tests. Thirty-two patients (89%) were hydrogen producers & 13 patients (36%) produced methane; 26 patients (72%) had whole blood thiamine levels below the lower limit of normal. CONCLUSION: This study supports the idea that gastric acid secretion prevents SIBO. Since 72% of GBS patients had low thiamine levels, micronutrient absorption is altered after GPS. Present physiological evidence supports the presence of a saturable, carrier-mediated exchange mechanism for thiamine transport across human jejunum; therefore, SIBO may alter this carrier-mediated mechanism.

Identification of Nuclear Import Mechanisms for the Neuronal Cdk5 Activator

The activation of Cdk5 by p35 plays a pivotal role in a multitude of nervous system activities ranging from neuronal differentiation to degeneration. A fraction of Cdk5 and p35 localizes in the nucleus where Cdk5-p35 exerts its functions via protein phosphorylation, and p35 displays a dynamic localization between the cytoplasm and the nucleus. Here, we examined the nuclear import properties of p35. In nuclear import assays, p35 was actively transported into the nuclei of digitonin-permeabilized HeLa cells and cortical neurons by cytoplasmic carrier-mediated mechanisms. Importin-beta, importin-5, and importin-7 were identified to import p35 into the nuclei via a direct interaction with it. An N-terminal region of p35 was defined to interact with the above importins, serving as a nuclear localization signal. Finally, we show that the nuclear localization of p35 does not require the association of Cdk5. Furthermore, Cdk5 and importin-beta/5/7 are mutually exclusive in binding to p35. These results suggest that p35 employs pathways distinct from that used by Cdk5 for transport to the nucleus.

Study on crystalline properties of Er–Si–O compounds in relation to Er-related 1.54 μm photoluminescence and electrical properties

Er–Si–O crystalline compounds, which exhibit superlattice structures and sharp and strong Er-related 1.54 μm photoluminescence (PL) spectra at room temperature have been formed by self-assembling growth mechanism. Oxidation of the starting materials which have Si and Er at an atomic ratio of 2:1 are prepared and then oxidation and succeeding high-temperature annealing in Ar above 1250 °C cause a self-assembled superlattice-structured Er–Si–O crystalline compounds. The control of the ratio of Si and Er, as well as the following oxidation and annealing processes, is found to be sensitive to the crystalline properties, PL spectra and electrical properties. In this study, Er–Si–O crystalline thin films are formed on Si substrates by sol–gel and MOMBE methods, and their crystalline properties such as crystalline orientation and concentration ratio of Er, Si and O are investigated. Crystalline Er–Si–O films of high orientation are successfully grown on Si(1 0 0) and its inclined surface. The PL and excitation spectra, fluorescence decay and the electrical properties are found to be strongly related to the crystalline properties. Excess O causes a broader 1.54 μm PL spectra, slower fluorescence decay, lower carrier-mediated excitation and higher resistivity. A precise control of O is found to be necessary to grow superlattice-structured Er–Si–O compounds, which are semiconducting and are excitable via carrier-mediated excitation mechanism.

Epithelial Organic Cation Transporters Ensure pH-Dependent Drug Absorption in the Airway

Most inhaled beta(2)-adrenergic agonist and anticholinergic bronchodilators have low lipid solubility because of their transient or permanent positive net charge at physiologic pH. Airway absorption of these cationic drugs is incompletely understood. We examined carrier-mediated mechanisms of cationic drug uptake by human airway epithelia. Airway tissues and epithelial cells, obtained from lung donors without preexisting lung disease, were evaluated for organic cation transporter expression by quantitative RT-PCR and immunofluorescence. For in vitro functional studies on primary airway epithelial cells, uptake of the cationic fluorophore 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP+) was characterized. Quantitative RT-PCR analysis demonstrated high mRNA levels for two polyspecific organic cation/carnitine transporters, OCTN1 and OCTN2, in human airway epithelia. Immunofluorescence of human airway sections confirmed OCTN1/2 protein expression, with a predominant localization to the apical portion of epithelial cells. Primary airway epithelial cells showed a carrier-mediated, temperature-sensitive and saturable uptake of ASP(+). Seventy-five to eighty percent of ASP(+) uptake was inhibited by L-carnitine, an OCTN2-carried zwitterion. The uptake was pH dependent, with approximately 3-fold lower rates at acidic (pH 5.7) than at alkaline (pH 8.2) extracellular pH. Albuterol and formoterol inhibited ASP(+) uptake, suggesting that all these molecules are carried by the same transport mechanism. These findings demonstrate the existence and functional role of a pH-dependent organic cation uptake machinery, namely OCTN1 and OCTN2, in human airway epithelia. We suggest that epithelial OCTN1/2 are involved in the delivery of inhaled cationic bronchodilators to the airway tissue.

Interaction of Amphetamines and Related Compounds at the Vesicular Monoamine Transporter

Amphetamine-type agents interact with the vesicular monoamine transporter type 2 (VMAT(2)), promoting the release of intravesicular neurotransmitter and an increase in cytoplasmic neurotransmitter. Some compounds, such as reserpine, "release" neurotransmitter by inhibiting the ability of VMAT(2) to accumulate neurotransmitter in the vesicle, whereas other types of compounds can release neurotransmitter via a carrier-mediated exchange mechanism. The purpose of this study was to determine, for 42 mostly amphetamine-related compounds, their mode of interaction with the VMAT(2). We used a crude vesicular fraction prepared from rat caudate to assay VMAT(2) activity. Test compounds were assessed in several assays, including 1) inhibition of [(3)H]dihydrotetrabenazine binding, 2) inhibition of vesicular [(3)H]dopamine uptake, and 3) release of preloaded [(3)H]dopamine and [(3)H]tyramine. Several important findings derive from this comprehensive study. First, our work indicates that most agents are VMAT(2) substrates. Second, our data strongly suggest that amphetamine-type agents deplete vesicular neurotransmitter via a carrier-mediated exchange mechanism rather than via a weak base effect, although this conclusion needs to be confirmed via direct measurement of vesicular pH. Third, our data fail to reveal differential VMAT(2) interactions among agents that do and do not produce long-term 5-hydroxytryptamine depletion. Fourth, the data reported revealed the presence of two pools of [(3)H]amine within the vesicle, one pool that is free and one pool that is tightly associated with the ATP/protein complex that helps store amine. Finally, the VMAT(2) assays we have developed should prove useful for guiding the synthesis and evaluation of novel VMAT(2) agents as possible treatment agents for addictive disorders.

Transport of levovirin prodrugs in the human intestinal Caco-2 cell line

The transport of 10 amino acid ester prodrugs of levovirin (LVV) was investigated in the human intestinal Caco-2 cell line in order to overcome the poor oral bioavailability of LVV, an investigational drug for the treatment of hepatitis C infection. The prodrugs were designed to improve the permeability of LVV across the intestinal epithelium by targeting the di/tri-peptide carrier, PepT1. Caco-2 cell monolayers were employed to study the transport and hydrolysis properties of the prodrugs. Among all mono amino acid ester prodrugs studied, the LVV-5'-(L)-valine prodrug (R1518) exhibited the maximum increase (48-fold) in permeability with nearly complete conversion to LVV within 1 h. Di-amino acid esters did not offer significant enhancement in permeability comparing with mono amino acid esters and exhibited slower conversion to LVV in Caco2 cell monolayers. Pharmacokinetic screening studies of the prodrugs in rats yielded the highest fold increase (6.9-fold) of AUC with R1518 and in general displayed a similar trend to that observed in increases of permeability in Caco-2 cells. Mechanisms involved in the Caco-2 cell transport of R1518 were also investigated. Results of bi-directional transport studies support the involvement of carrier-mediated transport mechanisms for R1518, but not for the LVV-5'-(D)-valine prodrug or LVV. Moreover, the permeability of R1518 was found to be proton dependent. PepT1-mediated transport of R1518 was supported by results of competitive transport studies of R1518 with the PepT1 substrates enalapril, Gly-Sar, valganciclovir, and cephalexin. R1518 was also found to inhibit the permeability of valganciclovir and cephalexin. These results suggest that R1518 is a PepT1 substrate as well as an inhibitor.

Characteristics of Substance P Transport Across the Blood–Brain Barrier

Substance P (SP; NH3(+)-Arg(+)-Pro-Lys(+)-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) belongs to a group of neurokinins that are widely distributed in the central nervous system and peripheral nervous system. The biological effects mediated by SP in the central nervous system include regulation of affective behavior, emesis, and nociception. Many of these actions are believed to be the result of the binding of SP to the neurokinin-1 (NK-1) receptor and subsequent transport across the blood-brain barrier (BBB). The objective of the study was to investigate the involvement of the NK-1 receptor in the permeation of SP across the BBB. Transport of 3H SP (1-13 nM) was investigated using BBMEC monolayers grown on polycarbonate membranes mounted on a Side-bi-Side diffusion apparatus. 3H SP samples were analyzed by scintillation spectrometry. Liquid chromatography-tandem mass spectrometry was used to monitor the transport at higher concentrations (micromolar). SP transport across BBMEC monolayers was found to be saturable (Km = 8.57 +/- 1.59 nM, Vmax = 0.017 +/- 0.005 pmol min(-1) mg(-1) protein) in the concentration range of 0-13 nM. Significant (p < 0.05) decline in 3H SP permeation was observed in the presence of unlabeled SP and at 4 degrees C, indicating that the transport process is carrier-mediated. High-performance liquid chromatography analysis showed no significant metabolism of 3H SP in either the donor or receiver chambers. 3H SP transport was inhibited by 2-11 SP (p < 0.05) but not by any other fragments, indicating that both the C- and N-terminal regions are essential for molecular recognition by the receptor. Endocytic inhibitors (chloroquine, phenylarsine oxide, monensin, and brefeldin) did not inhibit SP transport, suggesting the involvement of a nonendocytic mechanism in SP permeation. Pro(9) SP, a high-affinity substrate for the NK-1 major subtype receptor, significantly (p < 0.05) inhibited the transport of SP. However, Sar(9)Met(O2)(11) SP, a high-affinity substrate for the NK-1 minor subtype receptor, septide, and neurokinin A, inhibitors of NK-1 and neurokinin-2 (NK-2) receptors, respectively, did not produce any inhibition of SP transport. Western blot analysis confirmed the presence of the NK-1 receptor in BBMEC monolayers. The above results provide functional and molecular evidence for the existence of a carrier-mediated mechanism in the transport of SP across the BBB. The effects of specific inhibitors and the results of Western blot analyses demonstrate the involvement of the NK-1 receptor in the transport of SP across the BBB.

The complexity of intestinal absorption and exsorption of digoxin in rats

The potential multiple carrier-mediated mechanisms involved in the transport of digoxin in rat intestine were investigated by the rapid filtration method in rat intestinal brush-border vesicles (BBMV) and in vitro Ussing chambers. The uptake of digoxin showed a typical overshoot phenomenon in the presence of an inward proton gradient and an outward bicarbonate gradient, or an outward glutathione gradient in BBMV. Good fitting to an equation consisting of both saturable and linear terms was obtained using non-linear regression analysis. GF120918, a specific P-gp inhibitor, significantly increased the absorptive permeability of digoxin in rat ileum (7.02 × 10 −7 cm/s versus 2.11 × 10 −6 cm/s with GF120918) but the addition of DIDS (0.5 mM), an anionic transporter inhibitor, or bromosulfophthalein (0.1 mM), an Oatp inhibitor, in the presence of GF120918 decreased the absorptive permeability compared with GF120918 alone (2.11 × 10 −6 cm/s versus 1.46 × 10 −6 cm/s, p < 0.01 and 2.11 × 10 −6 cm/s versus 1.60 × 10 −6 cm/s, p < 0.05, respectively). The above results suggest the involvement of carrier-mediated uptake mechanism, possibly Oatp, in digoxin absorption. Interestingly, GF120918 (1 μM) did not abolish the polarized transport of digoxin in rat jejunum and ileum, and DIDS (0.5 mM), not a P-gp inhibitor, and MK571 (50 μM), an MRP-selective inhibitor, can also significantly decrease the exsorptive permeability of digoxin. This result indicates the involvement of non-P-gp efflux transporter in digoxin secretion and this transporter is DIDS and MK571-sensitive. Contrary to conventional concept, our studies show that intestinal absorption of digoxin may involve both active uptake and efflux transporters. Our study may have clinical implications in drug–drug or drug–food interactions involving transporters.

Dilute magnetic oxides

Dilute magnetic oxides are transparent, wide-bandgap materials that behave ferromagnetically when doped with a few percent of a magnetic 3d cation. The magnetism, which appears well below the cation percolation threshold, cannot be understood in terms of the conventional theory of magnetism in insulators; nor can a carrier-mediated ferromagnetic exchange mechanism account for the magnitude of the Curie temperatures, which are well in excess of 400 K. The phenomenon is observed in thin films and nanocrystals, but not in well-crystallized bulk material. Experimental artefacts and segregation of secondary ferromagnetic phases can explain some observations, but the existence of a novel type of magnetism related to defects other than the magnetic dopants is a likely inference from the data.

Identification and Functional Expression of a Carrier-Mediated Riboflavin Transport System on Rabbit Corneal Epithelium

Purpose: To investigate the functional expression of a carrier-mediated transport mechanism for riboflavin (vitamin B2) across cultured rabbit primary corneal epithelial cells (rPCECs) and intact rabbit cornea. The secondary objective was to understand the physiological significance behind the presence of such a transport system for riboflavin on the apical side of the corneal epithelium. Methods: rPCECs and freshly excised rabbit corneas were selected as in vitro and ex vivo models, respectively. Transport and uptake characteristics of [3H]riboflavin were determined at various time points, concentrations, temperatures, and pH. Substrate specificity, energy, and ion dependence studies were carried out to characterize the translocation mechanism. Rabbit tear analysis was done with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to understand the physiological relevance of this transporter. Results: The uptake process in rPCECs was found to be concentration dependent and saturable at higher concentrations. The process was also independent of pH, Na+, and Cl− but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition, whereas unrelated vitamins did not interfere with the process. Transport of [3H]riboflavin across rabbit cornea was also saturable at higher concentration and energy dependent but Na+ independent. Substrate specificity studies across intact rabbit cornea produced results similar to the uptake studies in cultured rPCECs. LC-MS/MS analysis of rabbit tears showed the presence of riboflavin. Conclusions: Results suggest the presence of a specialized, high-affinity transport mechanism for riboflavin that is expressed on the apical side of rabbit corneal epithelium and may in turn be responsible for influx of riboflavin from tears to cornea.

Intestinal permeability of metformin using single-pass intestinal perfusion in rats

To characterize the intestinal transport and mechanism of metformin in rats and to investigate whether or not metformin is a substrate for P-glycoprotein (P-gp). The effective intestinal permeability of metformin was investigated using single-pass intestinal perfusion (SPIP) technique in male Waster rats. SPIP was performed in three isolated intestinal segments (duodenum, jejunum and ileum) at the same concentration of metformin (50 microg/mL) to test if the intestinal transport of metformin exhibited site-dependent changes, and in a same isolated intestinal segment (duodenal segment) at three different concentrations of metformin (10, 50, 200 microg/mL) to test if the intestinal transport of metformin exhibited concentration-dependent changes. Besides, P-gp inhibitor verapamil (400 microg/mL) was co-perfused with metformin (50 microg/mL) in the duodenum segment to find out if the intestinal absorption of metformin was affected by P-gp exiting along the gastrointestinal track. Stability studies were conducted to ensure that the loss of metformin could be attributed to intestinal absorption. The effective permeability values (P(eff)) of metformin in the jejunum and ileum at 50 microg/mL were significantly lower than those in the duodenum at the same concentration. Besides, P(eff) values in the duodenum at high concentration (200 microg/mL) were found to be significantly lower than those at low and medium concentrations (10 and 50 microg/mL). Moreover the co-perfusion with verapamil did not increase the P(eff) value of metformin at 50 microg/mL in the duodenum. Metformin could be absorbed from the whole intestine, with the main absorption site at duodenum. This concentration-dependent permeability behavior in the duodenum indicates that metformin is transported by both passive and active carrier-mediated saturable mechanism. The P(eff) value can not be increased by co-perfusion with verapamil, indicating that absorption of metformin is not efficiently transported by P-gp in the gut wall. Furthermore metformin is neither a substrate nor an inducer of P-gp. Based on the P(eff) values obtained in the present study and using established relationships, the human fraction dose absorbed for metformin is estimated to be 74%-90% along human intestine.