Saturable Transport Mechanism Research Articles

Identification of transport systems involved in eflornithine delivery across the blood-brain barrier.

Human African Trypanosomiasis (HAT) is a neglected parasitic disease that continues to persist in sub-Saharan Africa. It is fatal if untreated. The first stage of the disease is associated with the presence of the parasite in the periphery and the second stage with the presence of the parasites in the CNS. The treatment of CNS stage HAT requires the drugs to cross the blood-brain barrier (BBB). Eflornithine is an amino acid analogue that is used to treat second stage HAT gambiense both alone and in combination with nifurtimox. Recent studies have identified that accumulation of eflornithine into the parasites (trypanosomes) involves the amino acid transporter (Trypanosoma brucei AAT6). In this study we tested the hypothesis that eflornithine uses a cationic amino acid transport system to cross the BBB. We particularly focused on system-y+ and system-B0,+. To do this we utilized specialist databases to compare the physicochemical characteristics of relevant molecules and an in vitro model of the BBB to explore the mechanisms of eflornithine delivery into the CNS. Our results confirmed that eflornithine is related to the endogenous amino acid, ornithine. At pH 7.4, eflornithine is predominately (92.39%) a zwitterionic (dipolar) amino acid and ornithine is predominately (99.08%) a cationic (tripolar) amino acid. In addition, the gross charge distribution at pH 7.4 of eflornithine is much smaller (+0.073) than that of ornithine (+0.99). Further results indicated that eflornithine utilized a saturable transport mechanism(s) to cross the hCMEC/D3 cell membranes and that transport was inhibited by the presence of other amino acids including ornithine. Eflornithine transport was also sodium-independent and sensitive to a y+-system inhibitor, but not a B0,+-system inhibitor. Eflornithine transport was also inhibited by pentamidine, suggestive of transport by organic cation transporters (OCT) which are expressed in this cell line. We confirmed expression of the y+-system protein, CAT1, and the B0,+-system protein, ATB0,+, in the hCMEC/D3 cells. We conclude that eflornithine uses the cationic amino acid transporter, system y+, and OCT to cross the BBB. This research highlights the potential of system-y+ to deliver drugs, including eflornithine, across the BBB to treat brain diseases.

A Na+-Dependent Mechanism Is Involved in Mucosal Uptake of Cinnamic Acid across the Jejunal Brush Border in Rats

Phenolic acids are present in all plant-derived foods and in most diets. Indirect evidence indicates substantial absorption of phenolic monomers from the gastrointestinal tract. However, the mechanisms involved in the absorptive process are unknown. The present study investigates mucosal uptake of radioactively labeled cinnamic acid as a model substance for monomeric cinnamic acid derivatives (e.g., cinnamic, ferulic or caffeic acid) in the rat jejunum using an in vitro mucosal uptake technique. The results indicate the existence of a Na+-dependent saturable transport mechanism for uptake of cinnamic acid across the jejunal brush border membrane. The observed Na+ dependence of jejunal cinnamate uptake seems not to be related to the activity of the Na+, H+ exchanger. Lowering the pH of the incubation medium resulted in a pronounced increase in mucosal cinnamate uptake that can be only partially explained by an increase in nonionic diffusion of cinnamic acid. Furthermore, jejunal uptake of cinnamate seems to be influenced by intracellular HCO3- and/or pH, since the addition of methazolamide to a HCO3- and CO2-free incubation medium significantly inhibited mucosal cinnamate uptake, whereas methazolamide was without an effect in the presence of HCO3- and CO2 in the incubation medium. Unlabeled cinnamic and ferulic acid as well as short-chain fatty acids (acetic, propionic and butyric acid) significantly inhibited Na+-dependent uptake of radioactively labeled cinnamic acid. Taken together, the results indicate the involvement of a Na+-dependent, carrier-mediated transport process in the uptake of cinnamic acid and structurally related substrates such as ferulic acid across the brush border membrane of rat jejunum.

Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice.

Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

The effects of both age and sex on irisin levels in paired plasma and cerebrospinal fluid in healthy humans

BackgroundAdipo-myokine irisin has important effects on the metabolism and functioning of multiple tissues and organs. However, the effects of aging and sex on irisin levels in cerebrospinal fluid (CSF) and on circulation have not been comprehensively studied. ObjectiveTo investigate whether aging and sex can affect irisin levels in both CSF and plasma; to determine whether CSF irisin uptake involves a saturable transport mechanism. Design and methodsIn the present study, the irisin levels in paired CSF and plasma samples drawn from 71 healthy individuals were used to investigate effects by using commercial ELISA kits and mass spectrometry. ResultsMultiple linear regression analysis results showed that CSF irisin levels are positively correlated with the CSF/plasma irisin ratio and age and that these levels present a reverse correlation with BMI. Age-related increases in CSF levels are validated by using ELISA and mass spectrometry. Higher plasma irisin levels are observed in men than women. CSF and plasma irisin levels are nonlinearly associated with the CSF/plasma irisin ratio, BMI, age and F scores. The CSF/plasma irisin ratio is U-shaped and associated with age. ConclusionsThere might be an age-related increase in irisin levels in the cerebrospinal fluid of healthy humans. Circulating irisin levels are higher in males than in females in the healthy population. A saturable mechanism might be involved in mediating the transport of circulating irisin across the blood-brain barrier. Factors shaping irisin levels for both circulation and the CSF of healthy humans must be further defined in future experiments.

Proopiomelanocortin, agouti-related protein, and leptin in human cerebrospinal fluid: correlations with body weight and adiposity.

Leptin and its neuronal targets, which produce proopiomelanocortin (POMC) and agouti-related protein (AgRP), regulate energy balance. This study characterized leptin, POMC, and AgRP in the cerebrospinal fluid (CSF) of 47 healthy human subjects, 23 lean and 24 overweight/obese (OW/OB), as related to BMI, adiposity, plasma leptin, soluble leptin receptor (s-OB-R), and insulin. POMC was measured since the POMC prohormone is the predominant POMC peptide in CSF and correlates with hypothalamic POMC in rodents. Plasma AgRP was similarly characterized. CSF leptin was 83-fold lower than in plasma and correlated strongly with BMI, body fat, and insulin. The relative amount of leptin transported into CSF declined with increasing BMI, ranging from 4.5 to 0.52%, consistent with a saturable transport mechanism. CSF sOB-R was 78-fold lower than in plasma and correlated negatively with plasma and CSF leptin. CSF POMC was higher in lean vs. OW/OB subjects (P < 0.001) and correlated negatively with CSF leptin (r = -0.60, P < 0.001) and with plasma leptin, insulin, BMI, and adiposity. CSF AgRP was not different in lean vs. OW/OB; however, plasma AgRP was higher in lean subjects (P = 0.001) and correlated negatively with BMI, adiposity, leptin, insulin, and HOMA (P < 0.005). Thus, CSF measurements may provide useful biomarkers for brain leptin and POMC activity. The striking negative correlation between CSF leptin and POMC could be secondary to leptin resistance and/or neuronal changes associated with obesity but may also indicate that POMC plays a primary role in regulating body weight and adiposity. The role of plasma AgRP as a neuroendocrine biomarker deserves further study.

Cellular Uptake of the Antitumor Agent Dp44mT Occurs via a Carrier/Receptor-Mediated Mechanism

The chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) shows potent and selective anticancer and antimetastatic activity. However, the mechanism by which it is initially transported into cells to induce cytotoxicity is unknown. Hence, the current investigation examined the cellular uptake of ¹⁴C-Dp44mT relative to two structurally related ligands, namely the aroylhydrazone ¹⁴C-pyridoxal isonicotinoyl hydrazone (¹⁴C-PIH) and the thiosemicarbazone (¹⁴C-2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (¹⁴C-Bp4eT). In marked contrast to the cellular uptake of ¹⁴C-PIH and ¹⁴C-Bp4eT, which were linear as a function of concentration, ¹⁴C-Dp44mT uptake was saturable using SK-N-MC neuroepithelioma cells (Bmax, 4.28 × 10⁷ molecules of chelator/cell; and Kd, 2.45 μM). Together with the fact that ¹⁴C-Dp44mT uptake was temperature-dependent and significantly (P < 0.01) decreased by competing unlabeled Dp44mT, these observations indicated a saturable transport mechanism consistent with carrier/receptor-mediated transport. Other unlabeled ligands that shared the saturated N4 structural moiety with Dp44mT significantly (P < 0.01) inhibited ¹⁴C-Dp44mT uptake, illustrating its importance for carrier/receptor recognition. Nevertheless, unlabeled Dp44mT most markedly decreased (¹⁴C-Dp44mT uptake, demonstrating that the putative carrier/receptor shows high selectivity for Dp44mT. Interestingly, in contrast to ¹⁴C-Dp44mT, uptake of its Fe complex [Fe(¹⁴C-Dp44mT)₂] was not saturable as a function of concentration and was much greater than the ligand alone, indicating an alternate mode of transport. Studies examining the tissue distribution of ¹⁴C-Dp44mT injected intravenously into a mouse tumor model demonstrated the ¹⁴C label was primarily identified in the excretory system. Collectively, these findings examining the mechanism of Dp44mT uptake and its distribution and excretion have clinical implications for its bioavailability and uptake in vivo.

The Fusion Protein of IFN-α and Apolipoprotein A-I Crosses the Blood–Brain Barrier by a Saturable Transport Mechanism

IFN-α is widely used for the treatment of chronic viral hepatitis and malignancies. However, systemic IFN-α treatment causes severe neuropsychiatric complications in humans, including depression, anxiety, and cognitive impairments. We have previously reported that the fusion protein formed by IFN-α and apolipoprotein A-I (IA) circulates bound to high-density lipoproteins (HDLs) and exhibits liver targeting, increased half-life, enhanced immunostimulatory activity, and reduced cytotoxicity. As the transport of HDLs across the blood-brain barrier is a highly complex and regulated process, in this study, we examine the effects of IA on the brain. Determination of IFN-α in brain and serum after hydrodynamic administration of different doses of a plasmid encoding IFN-α or IA showed that IA penetrated into the brain by a saturable transport mechanism. Thus, at high serum levels of the transgenes, the induction of IFN-sensitive genes and the number of phospho-STAT1(+) cell nuclei in the brain were substantially higher with IFN-α than with IA. This was associated with attenuation of neurodepression in mice given IA, as manifested by shorter immobility time in the tail suspension test. However, when given low doses of rIFN-α or the same antiviral units of HDLs containing IA, the induction of IFN-stimulated genes in the brain was significantly greater with the latter. In conclusion, IA crosses the blood-brain barrier not by diffusion, as is the case of IFN-α, but by a facilitated saturable transport mechanism. Thus, linkage to apolipoprotein A-I may serve to modulate the effects of IFN-α on the CNS.

Ovine Proinflammatory Cytokines Cross the Murine Blood-Brain Barrier by a Common Saturable Transport Mechanism

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with ¹³¹I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine ¹³¹I-IL-1β and ¹³¹I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine ¹³¹I-IL-1β or ¹³¹I-IL-6, respectively, across the BBB. Transport of both ovine ¹³¹I-IL-1β and ¹³¹I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine ¹³¹I-IL-1β or ¹³¹I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

Where Does Insulin Resistance Start?

During the last two decades, many studies have focused on the pivotal role of the hypothalamus in the control of energy metabolism (1). Hypothalamic nuclei, particularly the arcuate and the ventromedial, receive numerous peripheral inputs from adipokines (leptin, adiponectin, and resistin) and free fatty acids. These signals of “nutrient abundance” lead to the activation of multiple hypothalamic pathways that overlap and generate a plethora of central and peripheral responses (2). In this context, the central neuronal signals generated by insulin have been the most extensively studied (3). The pivotal experiment performed by Woods et al. (4) in the late 1970s showed that intracerebral infusion of insulin in baboons reduced food intake and body weight and paved the way for intense scientific investigation over subsequent years by highlighting the key role of insulin at the central nervous system (CNS) level. Insulin receptors (5) and components of the insulin signaling pathway (6) are widely distributed in the brain. Insulin interacts with its binding sites by crossing the blood-brain barrier through a receptor-mediated and saturable transport mechanism (7), although it has been hypothesized that insulin is also synthesized in the brain (7). Considerable evidence has been generated to indicate that insulin can modulate the expression of neuropeptides involved in the regulation of food intake within the CNS (8) and also can influence glucoregulation via CNS connections that regulate hepatic glucose production (9), glycogen synthesis in the skeletal muscle (10), and fat metabolism in adipocytes (11). By activating its receptors, insulin directly suppresses prepro-NPY mRNA transcription in the arcuate nucleus, leading to a reduction in NPY and a decrease in food intake (12), whereas intracerebroventricular delivery of insulin increases the expression of pro-opiomelanocortin (POMC) (13) (Fig. 1). This hypothalamic mode of insulin action resembles the well-known effect of leptin, suggesting that these …

Expression of the gas-transporting proteins, Rh B glycoprotein and Rh C glycoprotein, in the murine lung.

A family of gas-transporting proteins, the Mep/Amt/Rh glycoprotein family, has been identified recently. These are integral membrane proteins, are widely expressed in sites of gas transport, and are known to transport the gaseous molecule, NH(3), and recent evidence indicates they can transport CO(2). Because the mammalian lung is a critical site for gas transport, the current studies examine the expression of the nonerythroid members of this extended family, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in the normal mouse lung. Real-time RT-PCR and immunoblot analysis demonstrated both Rhbg and Rhcg mRNA and protein expression, respectively. Immunohistochemistry demonstrated both Rhbg and Rhcg were expressed in bronchial and bronchiolar epithelial cells. Rhbg was expressed by Clara cells, specifically, whereas all bronchial/bronchiolar epithelial cells, with the exception of goblet cells, expressed Rhcg. Rhbg expression was basolateral, whereas Rhcg exhibited apical and intracellular immunolabel, polarized expression similar to that observed in Rhbg- and Rhcg-expressing epithelial cells in other organs. There was no detectable expression of either Rhbg or Rhcg in alveolar endothelial or epithelial cells, in pneumocytes or in vascular tissue. In vitro studies using cultured bronchial epithelial cells confirm Rhbg and Rhcg expression, demonstrate that saturable, not diffusive, transport is the primary mechanism of ammonia/methylammonia transport, and show that the saturable transport mechanism has kinetics similar to those demonstrated previously for Rhbg and Rhcg. These findings suggest Rhbg and Rhcg may contribute to bronchial epithelial cell ammonia metabolism and suggest that they do not contribute to pulmonary CO(2) transport.

In vitro and in vivo pharmacokinetic characterization of two novel prodrugs of zidovudine

This work deals with the in vitro and in vivo pharmacokinetic characterization of 3′-azido-2′,3′-dideoxy-5′- O-oxalatoylthymidine (AZT-Ac) and 3′-azido-2′,3′-dideoxy-5′- O-isonicotinoyl-thymidine (AZT-Iso), two novel prodrugs of the anti-HIV agent zidovudine [3′-azido-2′,3′-dideoxythymidine (AZT)]. AZT, AZT-Ac and AZT-Iso intestinal permeation properties and plasma concentration profiles in rats after intravenous administration were studied. Using the everted gut sac intestinal permeation assay, it was observed that AZT was subjected to saturable transport mechanisms in the jejunum and the proximal ileum, while no saturation was found in the distal ileum. AZT-Ac was able to permeate the intestinal segment at a lower rate than AZT but resisting enzymatic hydrolysis, while no evidence of saturation was found. On the other hand, AZT-Iso was completely hydrolyzed in the intestinal tissue, with AZT being found in the permeated samples. In vivo studies demonstrated that AZT plasma half-life ( t 1/2) is extended after administration of AZT-Ac compared to AZT (2.16 and 0.96 h, respectively), while after administering AZT-Iso the t 1/2 of the regenerated AZT was shorter (0.38 h). A relationship is proposed between these observed in vivo pharmacokinetic features and previous studies of protein-binding properties, concluding that AZT-Ac is a very promising prodrug of AZT in the search for more effective and safer anti-HIV agents.

Muscarinic receptor regulation of osmosensitive taurine transport in human SH‐SY5Y neuroblastoma cells

The ability of G‐protein‐coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH‐SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions taurine influx was mediated exclusively by a Na+‐dependent, high‐affinity (Km=2.5 μM) saturable transport mechanism (Vmax=0.087 nmol/mg protein/min). Reductions in osmolarity of &gt;20% (under conditions of constant NaCl) resulted in an inhibition of taurine influx (&gt;30%) that could be attributed to a reduction in Vmax, whereas the Km for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine‐M (Oxo‐M), also resulted in an attenuation of taurine influx (EC50~0.7 μM). Although Oxo‐M‐mediated inhibition of taurine uptake could be observed under isotonic conditions (~25‐30%), the magnitude of inhibition was significantly enhanced by hypotonicity (~55‐60%), a result that also reflected a reduction in the Vmax, but not the Km, for taurine transport. Oxo‐M‐mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca2+ but was independent of protein kinase C activity. These results suggest that taurine uptake is reduced upon muscarinic receptor activation in a volume‐dependent fashion, thus facilitating taurine depletion from cells upon exposure to hypotonicity. Supported by NS23831 (SKF) GM007767 (DJF) and DK64959 (RB).

Carnitine/Organic Cation Transporter OCTN2 (Slc22a5) Is Responsible for Renal Secretion of Cephaloridine in Mice

Carnitine/organic cation transporter (OCTN) 2 (SLC22A5) plays a pivotal role in renal tubular reabsorption of carnitine, a vitamin-like compound, on apical membranes of proximal tubules, but its role in relation to therapeutic drugs remains to be clarified. The purpose of the present study was to elucidate the involvement of OCTN2 in renal disposition of a beta-lactam antibiotic, cephaloridine (CER), based on experiments with juvenile visceral steatosis (jvs) mice, which have a functional deficiency of the octn2 gene. Renal clearance of CER during constant intravenous infusion in wild-type mice was much higher than could be accounted for by glomerular filtration, but was decreased by increasing the infusion rate with minimal change in kidney-to-plasma concentration ratio, suggesting the existence of saturable transport mechanism(s) across the apical membranes. The plasma concentration profile and kidney-to-plasma concentration ratio after intravenous injection in jvs mice were higher than those in wild-type mice, whereas renal clearance in jvs mice was much lower than that in wild-type mice and could be accounted for by glomerular filtration. Uptake of CER by mouse OCTN2 was shown in Xenopus laevis oocytes expressing mouse OCTN2. The CER transport by OCTN2 exhibited saturation with K(m) of approximately 3 mM, which is similar to the renal CER concentration exhibiting saturation in renal clearance in vivo. The OCTN2-mediated CER transport was inhibited by carnitine and independent of Na(+) replacement in the medium. These results show OCTN2 on apical membranes of proximal tubules plays a major role in renal secretion of CER in mice.

Gabapentin Extended Release for the Treatment of Painful Diabetic Peripheral Neuropathy

A randomized, double-blind, placebo-controlled study was conducted in 147 patients to determine the efficacy and safety of gabapentin extended release (g-ER) in treating pain associated with diabetic peripheral neuropathy (DPN). Gabapentin, an immediate-release formulation, has demonstrated clinical efficacy in DPN patients but also a relatively high incidence of somnolence and dizziness at the doses required for effective treatment of DPN pain (1,2). Using the polymer-based AcuForm technology, g-ER tablets have been formulated to handle the saturable transport mechanism responsible for the lack of dose proportionality seen with gabapentin. When administered with a meal, g-ER tablets gradually expand and release the drug to the upper gastrointestinal …

Identification and Characterization of 3-Iodothyronamine Intracellular Transport

3-Iodothyronamine (T(1)AM) is a naturally occurring thyroid hormone metabolite with distinct biological effects that are opposite those of thyroid hormone. The known molecular targets of T(1)AM include both plasma membrane and intracellular proteins, suggesting that intracellular transport of T(1)AM may be an important component of its action, although no uptake mechanism has yet been described. Using various human cell lines, we show that, indeed, cellular uptake of T(1)AM occurs in multiple cell types and that this process involves specific, saturable, and inhibitable transport mechanisms. These mechanisms are sodium and chloride independent, pH dependent, thyronamine specific, and do not involve the likely candidate transporters of other monoamines, organic cations, or thyroid hormones. A large-scale RNA interference screen targeting the entire solute carrier superfamily of transporter genes reveals that the transport of T(1)AM into cells involves multiple transporters, and we identify eight transporters that may contribute to the uptake of T(1)AM in HeLa cells. This type of transporter small interfering RNA screening approach can be used in general to identify the constellation of transporters that participate in the intracellular disposition of compounds.

Muscarinic receptor regulation of osmosensitive taurine transport in human SH‐SY5Y neuroblastoma cells

The ability of G protein-coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH-SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1-100 microM), taurine influx was mediated exclusively by a Na(+)-dependent, high-affinity (K(m) = 2.5 microM) saturable transport mechanism (V(max) = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V(max), whereas the K(m) for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), also resulted in an attenuation of taurine influx (EC(50) approximately 0.7 microM). Although Oxo-M-mediated inhibition of taurine uptake could be observed under isotonic conditions (approximately 25-30%), the magnitude of inhibition was significantly enhanced by hypotonicity (approximately 55-60%), a result that also reflected a reduction in the V(max), but not the K(m), for taurine transport. Oxo-M-mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca(2+) but was independent of protein kinase C activity. In addition to Oxo-M, inclusion of either thrombin or sphingosine 1-phosphate also attenuated volume-dependent taurine uptake. The ability of Oxo-M to inhibit the influx of taurine was attenuated by 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid, an inhibitor of the volume-sensitive organic osmolyte and anion channel. 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid also prevented receptor-mediated changes in the efflux and influx of K(+) under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume-dependent efflux and uptake of taurine and that these events may be functionally coupled.

Hyperinsulinemia Rapidly Increases Human Muscle Microvascular Perfusion but Fails to Increase Muscle Insulin Clearance

Transport of insulin from the central circulation into muscle is rate limiting for the stimulation of glucose metabolism. By recruiting muscle microvasculature, insulin may promote its own movement into muscle interstitium. We tested whether in humans, as in the rat, insulin exerts an early action to recruit microvasculature within skeletal muscle. We further hypothesized that expansion of the microvascular volume of muscle would enhance muscle insulin clearance. Microvascular volume, total blood flow, and muscle insulin and glucose uptake (forearm balance method) were measured in 14 lean, healthy volunteers before and during a 2-h hyperinsulinemic-euglycemic clamp (1 mU x kg(-1) x min(-1)). Microvascular volume was measured using contrast-enhanced ultrasound. Forearm muscle microvascular volume increased within 20 min of insulin infusion (P < 0.01), whereas an effect to increase total forearm flow was not observed until 100 min. Forearm insulin uptake increased with physiological hyperinsulinemia (15 +/- 3 and 87 +/- 13 fmol x min(-1) x 100 ml(-1) basal vs. last 40 min of clamp, P < 0.001). However, the extraction fraction and clearance of insulin declined (P = 0.02, for each), indicating saturability of muscle insulin uptake at physiological hyperinsulinemia. Skeletal muscle contributes to peripheral insulin clearance both in the basal state and with physiological hyperinsulinemia. Insulin promptly expands human muscle microvascular volume but only slowly increases blood flow. Despite increased microvascular volume available for insulin uptake, muscle insulin clearance decreases significantly. These findings are consistent with the presence of a saturable transport mechanism facilitating the transendothelial transport of insulin into human muscle.

Tissue uptake of thyroid hormone by amino acid transporters

Thyroid hormones (THs) -- thyroxine (T4) and tri-iodothyronine (T3) -- are iodinated derivatives of the amino acid tyrosine, which regulates growth, development and critical metabolic functions. THs are taken up by target cells and act at the genomic level via nuclear thyroid receptors. Saturable transport mechanisms mediate the greater part of TH movement across the plasma membrane. System L1 permease is a transporter of THs and amino acids in mammalian adipose tissue, placenta and brain. T(3) is also a substrate of a putative System T transporter, which is selective for aromatic amino acids. The activity and functional mechanisms of these transporters can be crucial to cells in determining both their hormone sensitivity and their responses to change in circulating hormone concentrations or availability of competing substrates (e.g. amino acids). TH transporters are potentially important pharmacological targets in the design of novel or improved therapies for thyroid-related disorders.

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

Intestinal uptake and biodistribution of novel polymeric micelles after oral administration

To determine the fate of polymeric micelles after oral administration, we investigated the possible transport of polymeric micelles across Caco-2 monolayers and their biodistribution in rats after per os administration of [ 14C]-labelled mmePEG 750P(CL-co-TMC) micelles containing risperidone (BCS Class II drug). mmePEG 750P(CL-co-TMC) was able to cross Caco-2 monolayer via a saturable transport mechanism. The oral bioavailability of the polymer was 40%. Polymeric micelles based on mmePEG 750P(CL-co-TMC) showed very low clearance by the reticuloendothelial system (RES) and a renal excretion. A sustained release of risperidone was observed.