Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-D-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate f lux and twoelectrode voltage clamp recording. EAAT5-mediated Lglutamate uptake is sodium-and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium-and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.The uptake of glutamate and other excitatory amino acids is mediated by a gene family of high affinity sodium-dependent transporters that includes four known mammalian subtypes; in humans, we have termed these glutamate transporters excitatory amino acid transporter (EAAT) 1 through 4 (1, 2). The transport of glutamate is driven by the cotransport of sodium ions and the countertransport of potassium ions down their electrochemical gradients, and recent studies suggest that this complex process involves the cotransport of protons as well (3-6). Because there is net inward movement of positive charge with the transport of each molecule of glutamate, the transport process is readily studied in the Xenopus oocyte expression system by observing the associated current. In addition to these transport currents, however, we have found that application of substrate to the transporter also gates an uncoupled, passive flux of chloride ions (2, 7). The relative magnitude of this associated chloride conductance varies with each cloned EAAT subtype; for EAAT1-EAAT3, the magnitude of the chloride current at physiological membrane potentials is similar to that of the electrogenic cotransport current, but the currents generated by EAAT4 are almost entirely due to the flux of chloride ions. In vivo, a glutamatedependent current that has a transporter-like pharmacology is carried largely by chloride ions in retinal cone (8) and rod (9) photoreceptors and bipolar cells (10). In bipolar cells, this chloride current has been proposed to mediate the cone component of the ON bipolar cell light response (10). Although the properties of EAAT4 are similar to the glu...
The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Mü ller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Mü ller cells and the glutamateelicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells. Key words: glutamate transporter; uptake; amino acid; retina; chloride; molecular cloning; photoreceptors; Mü ller cells; bipolar cells; immunohistochemistryGlutamate is the predominant excitatory neurotransmitter in the vertebrate retina and is released from photoreceptors, bipolar cells, and ganglion cells (for review, see Massey, 1990). Termination of glutamatergic synaptic transmission requires uptake of glutamate by means of plasma membrane glutamate transporters, which may be present presynaptically and postsynaptically as well as in neighboring glial cells. These transporters are of particular importance in the retina, where glutamate is released continuously and at a high rate in the dark.Electrophysiological studies performed primarily in the salamander retina have demonstrated the existence of glutamate transporters in retinal glial (Müller) cells (Brew and Attwell,
Application of L-glutamate to retinal glial (Miiller) Fig. 2), gluconate salts were substituted. In solutions containing other anions (SCN-, C104, NO3, and I-), NaCI was replaced by the appropriate sodium salt. For experiments with varying concentrations of NaNO3 (see Fig. 5
Nanoparticles are currently being investigated in a number of human clinical trials. As information on how nanoparticles function in humans is difficult to obtain, animal studies that can be correlative to human behavior are needed to provide guidance for human clinical trials. Here, we report correlative studies on animals and humans for CRLX101, a 20-to 30-nm-diameter, multifunctional, polymeric nanoparticle containing camptothecin (CPT). CRLX101 is currently in phase 2 clinical trials, and human data from several of the clinical investigations are compared with results from multispecies animal studies. The pharmacokinetics of polymer-conjugated CPT (indicative of the CRLX101 nanoparticles) in mice, rats, dogs, and humans reveal that the area under the curve scales linearly with milligrams of CPT per square meter for all species. Plasma concentrations of unconjugated CPT released from CRLX101 in animals and humans are consistent with each other after accounting for differences in serum albumin binding of CPT. Urinary excretion of polymer-conjugated CPT occurs primarily within the initial 24 h after dosing in animals and humans. The urinary excretion dynamics of polymer-conjugated and unconjugated CPT appear similar between animals and humans. CRLX101 accumulates into solid tumors and releases CPT over a period of several days to give inhibition of its target in animal xenograft models of cancer and in the tumors of humans. Taken in total, the evidence provided from animal models on the CRLX101 mechanism of action suggests that the behavior of CRLX101 in animals is translatable to humans.nanomedicine | clinical translation | interspecies scaling | pharmacodynamics | Nanoparticles
Camptothecin (CPT) is a potent broad-spectrum anticancer agent that acts through inhibition of topoisomerase 1. Clinical development of CPT was unsuccessful due to poor drug solubility, insufficient in vivo stability of the active form, and toxicity. In order to address these issues, a polymeric nanoparticle comprised of cyclodextrin-poly(ethylene glycol) copolymer (CDP) conjugated to CPT (CRLX101) has been developed and Phase 2 clinical studies are ongoing. Camptothecin is conjugated to the polymer in its active form at 10-12 wt.% loading. CRLX101 self-assembles in solution into nanoparticles with an apparent solubility increase of >1000-fold as compared to the parent drug camptothecin. Preclinical studies exhibited CRLX101 pharmacokinetics superior to the parent drug. Drug concentration in tumor relative to plasma and other major organs is consistent with the enhanced permeation and retention (EPR) anticipated from a nanoparticle. Significant anti-tumor activity was observed that is superior when compared to irinotecan across a broad range of xenograft models. Pharmacokinetic data are consistent with the prolonged half-life and increased AUC. The CRLX101 preclinical and clinical data confirm that CDP can address not only solubility, formulation, toxicity, and pharmacokinetic challenges associated with administration of CPT, but more importantly, can impart unique biological properties, that enhance pharmacodynamics and efficacy of camptothecin.
Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC 50s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC 50 for Lglutamate to activate the anion conductance, without affecting the EC50 for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.
Purpose: Increased tumor hypoxia and hence elevated hypoxia-inducible factor-1a (HIF1a) is thought to limit the efficacy of vascular endothelial growth factor (VEGF) pathway-targeting drugs by upregulating adaptive resistance genes. One strategy to counteract this is to combine antiangiogenic drugs with agents able to suppress HIF1a. One such possibility is the investigational drug CRLX101, a nanoparticle-drug conjugate (NDC) containing the payload camptothecin, a known topoisomerase-I poison.Experimental Design: CRLX101 was evaluated both as a monotherapy and combination with bevacizumab in a preclinical mouse model of advanced metastatic ovarian cancer. These preclinical studies contributed to the rationale for undertaking a phase II clinical study to evaluate CRLX101 monotherapy in patients with advanced platinum-resistant ovarian cancer.
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