Saturable Transport Research Articles

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76).

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.

Intestinal permeability of chlorpyrifos using the single-pass intestinal perfusion method in the rat

The intestinal transport of chlorpyrifos (CPF), an organothiophosphate pesticide, was investigated using the single-pass intestinal perfusion (SPIP) technique in male, Sprague–Dawley rats. SPIP was performed in each isolated region of the small intestine (i.e. duodenum, jejunum and ileum) with three concentrations of CPF (0.1, 2.0 and 10 μM) at a flow rate of 0.25 ml/min. Preliminary binding and stability studies were conducted to ensure that the loss of CPF in the SPIP study can be attributed to intestinal absorption. The effective permeability ( P eff) of CPF was determined for each segment and concentration. CPF exhibits a high intestinal permeability over the length of the small intestine indicative of compounds that are well absorbed. Decreases in permeability values at the highest CPF concentration studied in the duodenum and ileum suggest a saturable transport process. Based on these results, passive, transcellular diffusion dominates the intestinal transport mechanism of CPF, with a saturable transport process evident in the duodenum and ileum. The P eff of CPF is in the range of drugs with high intestinal permeability and high fraction of dose absorbed indicating that CPF readily crosses the intestine. The dependence of CPF's P eff on concentration in the duodenum and ileum suggests that CPF is transported by a combination of mechanisms across the intestine. Using established relationships, the human fraction dose absorbed for CPF was estimated to be >99%. The permeability values obtained from this study may be useful in models of exposure assessment.

Effect of endotoxin on expression of TNF receptors and transport of TNF-alpha at the blood-brain barrier of the rat.

The transport mechanism mediating brain uptake of tumor necrosis factor (TNF)-alpha has been studied. When (125)I-labeled rat TNF-alpha was used in internal carotid artery perfusions in rats, the cytokine showed transcytosis through the blood-brain barrier in intact form (permeability-surface area product 0.34 +/- 0.13 microl. min(-1). g(-1)). Uptake was inhibited by low nanomolar concentrations of unlabeled rat TNF-alpha. Human TNF-alpha, which does not interact with the p80 TNF receptor in rodents, showed no brain uptake. mRNA expression of both p60 and p80 receptors could be demonstrated in native brain microvessel preparations. These transcripts increased to 149% (p60) and 127% (p80) of control 4 h after a systemic immune stimulation (2 mg/kg bacterial endotoxin ip). Lipopolysaccharide treatment did not alter the rate of brain uptake of TNF-alpha measured between 4 and 24 h later. In conclusion, a receptor-mediated mechanism is responsible for the transcytosis of TNF-alpha. Saturable transport, requiring the p80 receptor, occurs at concentrations encountered under pathophysiological conditions and therefore constitutes a relevant mechanism of communication between the immune system and the brain.

Simulation of trichloroacetic acid kinetics in the isolated perfused rat liver using a biologically based kinetic model.

Trichloroacetic acid (TCA) is a contaminant of drinking water. It induces peroxisome proliferation in livers of rats and mice and is hepatocarcinogenic in the latter species. Previous experimental studies of the kinetics of TCA in the isolated perfused rat liver (IPRL) at two doses have been reported. To gain more insight into the mechanistic processes controlling TCA kinetics in the liver a biologically based kinetic (BBK) model for the IPRL was used to analyze the experimental data. The IPRL was exposed to 25, 250, or 1000 microM TCA for 2 h in a recirculating perfusion system. These doses were not cytotoxic. The BBK model simulated the TCA concentration in perfusion medium and liver, and the biliary excretion of TCA. Separate protein binding studies showed that over 90% of TCA was bound to albumin in the perfusion medium whereas binding in liver homogenate was much lower. Integrating the information on protein binding into the BBK model, the hepatic uptake of TCA and its biliary excretion could be fitted assuming asymmetrical saturable transport at the sinusoidal membrane and linear transport at the bile canalicular membrane. To validate the BBK model, additional washout experiments were conducted in which the perfusion medium was replaced with TCA-free medium after 30 min of exposure of the liver to 1000 microM TCA. This approach illustrates the usefulness of BBK modeling for analyzing experimental kinetic data and gaining insight in kinetic mechanisms controlling the behavior of a chemical in the liver.

Novel Mitochondrial Creatine Transport Activity: IMPLICATIONS FOR INTRACELLULAR CREATINE COMPARTMENTS AND BIOENERGETICS

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

The mechanisms of immune-to-brain communication in inflammation as a drug target.

There is considerable evidence that the peripheral immune system can signal the brain to elicit a sickness response during infection and inflammation. The induction of the sickness response involves the expression of proinflammatory cytokines such as interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6, both in the periphery and in the brain. The mechanisms by which peripheral cytokines can affect brain function have been the subject of much debate. The precise mechanisms by which cytokines signal the central nervous system (CNS) are unknown, but possibilities include: 1) the direct entry of cytokine into the brain across the blood-brain barrier by a saturable transport mechanism: 2) the interaction of cytokine with circumventricular organs such as the orgnum vasculosum of the lamina terminalis [OVLT] and area postrema, which lack the blood-brain barrier; and 3) activation of afferent neurons of the vagus nerve. Increasing evidence has suggested that the afferent vagus nerve is an important pathway for immune-to-brain communication. However, there are inconsistent findings for the involvement of the afferent vagus nerve in the mediation of transmitting inflammatory signals to the brain. Thus, we describe here the functional relevance of the vagal afferent nerve in mediating these effects. An understanding of the mechanisms involved in immune-to-brain communication should permit us to create new drugs as therapeutic targets to decrease sickness or promote recovery. This review focuses on recent discoveries of the multipathway mechanisms for the induction of sickness behavior mediated through neuroimmune interactions in the CNS.

Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells.

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller V(max) for iron uptake compared with control cell lines. One clone was further characterized and found to display an approximately 50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.

Age-dependent changes in cation transport in the chicken erythrocyte

Previous work has shown that the transport phenotype of chicken erythrocytes changes with the age of the chicken. Here, we report changes in the transport of choline and K + in erythrocytes from chickens at different developmental ages. The transport of choline in chicken erythrocytes was predominantly via saturable transport systems, was highest in erythrocytes from 1-day-old chickens and declined with chicken age when tested at 2 weeks of age and in mature chickens. Both K m and V max values for choline transport in chicken erythrocytes declined with chicken age. Similarly, the total unidirectional influx of K + was highest in erythrocytes from 1-day-old chickens and declined with chicken age, as did ouabain-sensitive K + influxes, which can be attributed to the Na +/K + pump. In isotonic conditions, bumetanide-sensitive K + influxes, which can be attributed to the Na +–K +–2Cl − cotransporter, were only measurable in erythrocytes from 1-day-old chickens. However, when stimulated by hypertonic conditions, bumetanide-sensitive K + influxes were essentially identical in erythrocytes from 1-day- and 2-week-old chickens but decreased in erythrocytes from mature chickens. We conclude that both choline and K + influxes decrease significantly in erythrocytes from chickens with increasing age. The changes are substantial but complex and may involve both regulation of existing transporters, and substitution or deletion of specific transporter isoforms.

New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle.

Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 +/- 9.4 microM and maximal velocity value 17.3 +/- 3.1 pmol x min(-1) x mg vesicle protein(-1)), which cotransports Cr into skeletal muscle together with Na(+) and Cl(-) ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and NH(2)-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of approximately 58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [(14)C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies.

In Vivo Measurement of Blood‐Brain Barrier Permeability

This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood-brain barrier. Methods for the measurement of drug influx and efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier-mediated or saturable transport.

Population pharmacokinetics and pharmacokinetic‐pharmacodynamic relationships of the novel anticancer agent E7070 in four phase I studies

E7070 is a novel chloroindolyl sulphonamide being developed as an anti‐cancer agent for the treatment of solid tumours. E7070 arrests cell cycle progression in the G1 phase and induces apoptosis. In various in vitro and in vivo models, E7070 appeared to be highly active. Four phase I dose‐escalation studies have been conducted to identify the dose‐limiting toxicities (DLTs) and the maximum tolerated dose. Adult patients with advanced solid tumours resistant or not amenable to conventional therapy received E7070 at four different treatment schedules: 1) 1 h i.v. infusion (doses 50–1000 mg m−2), 2) daily times five 1 h i.v. infusion (d×5) (10–200 mg m−2 day−1), 3) weekly times four 1 h infusion (40–500 mg m−2 week−1), 4) 120 h continuous i.v. infusion (civ) (6–200 mg m−2 day−1). DLTs were haematological in all studies. Pharmacokinetic (PK) sampling was performed in 143 patients (corresponding numbers of patients were 40, 35, 43 and 25) during the first cycle. In this study we investigated the PK and its relationship with the pharmacodynamics for all studies. A population PK model was developed to describe the combined data of the four studies. E7070 showed non‐linear PK at higher dose levels. Therefore, a basic 3‐compartmental model was extended with saturable transport to one of the peripheral compartments and parallel linear and a non‐linear pathways of elimination from the central compartment. The model adequately described the data. A validation using a bootstrap procedure indicated that the model was robust. Total clearance (CL) of E7070 was highly dependent on the plasma concentration; shortly after infusion, at the higher concentrations (i.e. 10–200 mg l−1), CL was 0.5 l h−1 and gradually increased to 5.5 l h−1 at the lower concentrations (0.1–0.01 mg l−1). For each individual, Bayesian estimates of AUC (area under the plasma concentration time curve) were obtained using the PK model. The percentage decreases in neutrophils (ANC) and thrombocytes (THR) were plotted against dose, dose level and AUC for each study. In general, AUC was a better predictor for haematological toxicity than dose and dose level, as variability was reduced when related to AUC. The relationship between % decrease in ANC and THR and AUC could be described using a maximum effect (Emax) model. A maximum % decrease in ANC and THR of approximately 95% was reached with all four schedules.In conclusion, the non‐linear PK of E7070 in all treatment schedules was adequately described using the population PK model. The dose‐limiting, haematological toxicity of E7070 is related to the total exposure, expressed as AUC.

Betaine is a highly effective organic osmolyte but does not appear to be transported by established organic osmolyte transporters in mouse embryos.

Betaine protects early preimplantation mouse embryos against increased osmolarity in vitro, functioning as an organic osmolyte. Betaine is effective at very low external concentrations, with half-maximal protection of 1-cell embryo development to blastocysts at approximately 50 microM, making it one of the best osmoprotectants for mouse preimplantation embryos. We performed studies designed to determine whether known high-affinity organic osmolyte transporters could account for the ability of betaine to act as an organic osmolyte in preimplantation embryos. We found no evidence in 1-cell embryos of transport by a betaine/GABA transporter (BGT1), the osmoregulated betaine transporter found in a number of cell types, as betaine and GABA did not inhibit each other's transport. Instead, all saturable GABA transport in embryos was apparently via the beta-amino acid transporter. We also found that the glycine transporter, GLY, which mediates osmoprotective transport of glycine in early preimplantation embryos, does not appear to transport betaine. Finally, increased osmolarity did not induce any detectable System A amino acid transporter activity, which is osmotically-inducible in other cells and can transport betaine. There does appear, however, to be a saturable betaine transporter in 1-cell mouse embryos, as considerable 14C-betaine transport was measured which was substantially inhibited by excess unlabeled betaine. Our data imply that betaine functions as an organic osmolyte in embryos due to its saturable transport via a mechanism distinct from known osmolyte transporters. We propose that an unidentified high-affinity betaine transporter may be expressed in early embryos and mediate transport of betaine as an organic osmolyte.

Functional impairment of renal organic cation transport in experimental diabetes.

This study was designed to determine the effect of diabetes on the function of the renal organic cation transport system that mediates the excretion of a wide variety of toxicants and drugs. The experiments compared the ability of renal cortex slices from streptozotocin-induced diabetic and non-diabetic rats to accumulate the model cation, 14C-tetraethylammonium under controlled conditions. Initial experiments demonstrated a progressive decline in tetraethylammonium accumulation with increasing duration of diabetes. The maximal decrease was observed at 21 days after streptozotocin injection. Time-dependent incubations revealed that tetraethylammonium uptake from both diabetic and non-diabetic rats followed a curvilinear pattern expected of an active process. However, at steady state the diabetic-derived slices accumulated a significant 38% less tetraethylammonium versus slices from non-diabetics. Concentration-dependent incubations of tetraethylammonium (0.01-10 mM, 60 min.) demonstrated saturable transport in both diabetic and non-diabetic slices with a significantly decreased capacity of diabetic-derived slices to accumulate tetraethylammonium. Cellular respiration rates in the two groups were not different. Insulin treatment of the diabetic rats prevented the transport decline. While the causative factor of the transport impairment in diabetes is unresolved, this study documents an aspect of diabetic nephropathy that has not been previously reported but which may have important implications for renal excretion of cationic drugs and toxicants. The results also provide a mechanism for the well-documented "protection phenomenon" by which the kidneys of diabetic rats are resistant to nephrotoxicity induced by the chemotherapeutic agent cisplatin.

Selected contribution: circadian rhythm of tumor necrosis factor-alpha uptake into mouse spinal cord.

Circadian variations in the actions of tumor necrosis factor-alpha (TNF-alpha) have been observed. Because a saturable transport system at the blood-brain barrier mediates most of the influx of TNF-alpha from blood to the central nervous system (CNS), the circadian variation of the CNS effects of TNF-alpha could be related to changes in this transport system. Accordingly, we measured the uptake of intravenously injected TNF-alpha into various CNS regions at different times and compared these measurements with the uptake into a peripheral control (muscle). We found that the spinal cord, but not the brain, showed a circadian rhythm in the uptake of TNF-alpha. This pattern is similar to that of leptin but different from that of interleukin-1. The circadian rhythm of the influx of TNF-alpha into this region of the CNS suggests a functional role for the spinal cord in the physiological actions of TNF-alpha.

Copper transport by lobster (Homarus americanus) hepatopancreatic mitochondria.

Mechanisms of copper transport into purified mitochondrial suspensions prepared from the hepatopancreas of the Atlantic lobster Homarus americanus were investigated. Mitochondria were purified by combining methods of differential and Percoll-gradient centrifugation, and copper transport was studied using the copper-sensitive fluorescent dye Phen Green. Copper transport by this mitochondrial preparation was kinetically the sum of saturable and non-saturable transfer components. Addition of 500 micromol x l(-1) Ca2+ or 500 nmol x l(-1) Ruthenium Red abolished the non-saturable copper transport component, significantly (P<0.01) reduced the apparent binding affinity of the saturable transport component, but was without effect (P>0.05) on the apparent maximal transport velocity of the saturable transfer process. The antiport inhibitor diltiazem (500 micromol x l(-1)) acted as a mixed inhibitor of the saturable transport mechanism, but had no effect on the non-saturable component of transfer. These results suggest that the non-saturable copper influx process was probably by way of the well-known Ruthenium-Red-sensitive Ca2+ uniporter and that the saturable transport component was probably due to a combination of both the Na+-dependent, diltiazem-sensitive 1Ca2+/2Na+ antiporter and the Na+-independent, diltiazem-insensitive 1Ca2+/2H+ antiporter. A model is discussed relating these mitochondrial copper uptake processes to the transfer of metal ions across the epithelial brush-border membrane.

PH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical neurons.

In this study, Zn(2+) transport in rat cortical neurons was characterized by successfully combining radioactive tracer experiments with spectrofluorometry and fluorescence microscopy. Cortical neurons showed a time-dependent and saturable transport of (65)Zn(2+) with an apparent affinity of 15-20 microM. (65)Zn(2+) transport was pH dependent and was decreased by extracellular acidification and increased by intracellular acidification. Compartmentalization of newly transported Zn(2+) was assessed with the Zn(2+)-selective fluorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn(2+). Depletion of intracellular Zn(2+) with the membrane-permeant chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) resulted in the complete loss of punctate zinquin labeling. After Zn(2+) depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 microM Zn(2+), pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 microM Zn(2+), pH 6.0. These data were confirmed in parallel (65)Zn(2+) transport experiments.

Insulin permeability across an in vitro dynamic model of endothelium.

Endothelium insulin permeability was investigated using in vitro, dynamic culture of endothelial cells. Endothelial cells were cultured in a hollow fiber apparatus and continuously exposed to a flow. Transendothelial electrical resistance and permeability to [14C]sucrose and [14C]inulin were used to monitor the integrity of the endothelial monolayer. Under these experimental conditions, measurements of insulin permeability, investigated at increasing hormone concentrations, suggested that the predominant transendothelial insulin fluxes were attributable to bidirectional convective transport rather than to a saturable transport mechanism, in agreement with in vivo experiment results published earlier. Analytical determinations of insulin catabolism demonstrated a low percent of insulin degradation by the endothelium, leading to production of insulin metabolites qualitatively identical to those produced by human monocytes. The findings of this paper indicated that (a) insulin crosses the endothelial monolayer by paracellular "leak" and endothelial insulin receptors have a minor (if any) role in insulin transport; (b) degradation of the hormone by BAEC is minimal; (c) the in vitro, dynamic culture of endothelial cells presented here should represent a valuable transport model system to study permeability mechanisms of insulin and many other drugs.

Interactions of glucagon-like peptide-1 (GLP-1) with the blood-brain barrier.

Glucagon-like peptide-1 (GLP-1) reduces insulin requirement in diabetes mellitus and promotes satiety. GLP-1 in the periphery (outside the CNS) has been shown to act on the brain to reduce food ingestion. As GLP-1 is readily degraded in blood, we focused on the interactions of [Ser8]GLP-1, an analog with similar biological effects and greater stability, with the blood-brain barrier (BBB). The influx of radiolabeled [Ser8]GLP-1 into brain has several distinctive characteristics: 1. A rapid influx rate of 8.867 +/- 0.798 x 10(4) mL/g-min as measured by multiple-time regression analysis after iv injection in mice. 2. Lack of self-inhibition by excess doses of the unlabeled [Ser8]GLP-1 either iv or by in situ brain perfusion, indicating the absence of a saturable transport system at the BBB. 3. Lack of modulation by short-term fasting and some other ingestive peptides that may interact with GLP-1, including leptin, glucagon, insulin, neuropeptide Y, and melanin-concentrating hormone. 4. No inhibition of influx by the selective GLP-1 receptor antagonist exendin(9-39), suggesting that the GLP-1 receptor is not involved in the rapid entry into brain. Similarly, there was no efflux system for [Ser8]GLP-1 to exit the brain other than following the reabsorption of cerebrospinal fluid (CSF). The fast influx was not associated with high lipid solubility. Upon reaching the brain compartment, substantial amounts of [Ser8]GLP-1 entered the brain parenchyma, but a large proportion was loosely associated with the vasculature at the BBB. Finally, the influx rate of [Ser8]GLP-1 was compared with that of GLP-1 in a blood-free brain perfusion system; radiolabeled GLP-1 had a more rapid influx than its analog and neither peptide showed the self-inhibition indicative of a saturable transport system. Therefore, we conclude that [Ser8]GLP-1 and the endogenous peptide GLP-1 can gain access to the brain from the periphery by simple diffusion and thus contribute to the regulation of feeding.

Food Deprivation Decreases Blood Galanin-Like Peptide and Its Rapid Entry into the Brain

Galanin-like peptide (GALP) was recently isolated from the hypothalamus, where its expression is influenced by leptin and food deprivation. Since leptin crosses the blood-brain barrier (BBB) by a saturable transport system that is downregulated by fasting, we examined the effect of leptin and fasting on the entry of GALP into mouse brain. Multiple-time regression analysis showed that the basal influx of ¹²⁵I-GALP from blood was rapid (K_i = 9.49 ± 0.72 × 10^–4 ml/g·min). This influx was not affected by leptin but was significantly decreased by food deprivation for 24 or 48 h, accompanied by decreased immunoreactive plasma GALP at 48 h, but not at 24 h. By contrast, pretreatment of mice fasted for 24 h with glucose resulted in a significant increase in the blood-to-brain influx of GALP that was not accompanied by increased immunoreactive plasma GALP. HPLC showed that most of the GALP crossed the BBB in an intact form, and capillary depletion studies showed that more than 93% of the GALP crossing entered the parenchyma of the brain rather than being bound to the endothelial cells of the capillaries composing the BBB or being reversibly associated with the vasculature. Efflux of ¹²⁵I-GALP occurred at the rate of the normal reabsorption of CSF, and the octanol-buffer partition coefficient showed insufficient lipophilicity to explain the fast rate of influx. When ¹²⁵I-GALP was perfused in blood-free buffer, the self-inhibition characteristic of a saturable transport system was evident even though capillary gel electrophoresis showed GALP aggregating as a trimer. Capillary zone electrophoresis showed protein binding of GALP in serum, perhaps facilitating its interactions at the BBB. In particular, these studies show for the first time (1) that immunoreactive GALP is present in blood where (2) its concentrations are reduced by food deprivation, and (3) that there is a rapid blood-to-brain influx of intact GALP (4) which is decreased by fasting and (5) increased by pretreatment with glucose.

Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: meaningful controls

Multiple-time regression analysis has been used to study the influx of radiolabeled peptides and polypeptides across the blood-brain barrier (BBB). This study used both tritiated and iodinated leptin to clarify several issues associated with these measurements. Recombinant murine leptin was radiolabeled with 3H by derivatization or with 125I by the iodobead method and each studied separately in mice. Intact 3H-leptin had a higher apparent influx rate from blood to brain than did intact 125I-leptin, correlating with its higher proportion of reversible association with the capillary lumen that would misleadingly appear to reflect entry. Yet the majority of 3H-leptin and 125I-leptin reached brain parenchyma. There was no significant difference in the influx rate between cerebral cortex and the subcortical regions, thus ruling out a predominant contribution of simple diffusion through the circumventricular organs or choroid plexuses outside the BBB. The influx of radiolabeled leptin, especially 125I-leptin, was decreased by excess unlabeled leptin, supporting the presence of a saturable transport system for leptin at the BBB. To identify the specificity of the transport system and determine whether it is shared by 3H-leptin and 125I-leptin, these radioactively labeled leptins were heat-denatured. Denaturation had no effect on the fast influx of 3H-leptin, but abolished the entry of 125I-leptin into brain; excess denatured leptin failed to inhibit the influx of either 3H-leptin or 125I-leptin. This indicates that the conformation of 125I-leptin is similar to that of native unlabeled leptin, so that iodination would be the better choice for investigating the interaction of leptin with the BBB. However, 3H-leptin can use the same transport system, as shown by inhibition of its influx by unlabeled leptin, whereas the derivatization procedure altered its biophysical properties such that its non-saturated influx was greatly enhanced. Finally, the rapid influx of radioactively labeled leptin contrasted greatly with that of the reference compounds 99mTc-albumin and 3H-inulin which had no significant penetration of the BBB. Thus, with additional considerations such as stability and interactions with the vasculature, multiple-time regression analysis is sensitive and selective for study of the penetration of peptides across the BBB.