The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine and, thus, in the termination of normal dopaminergic neurotransmission. DAT is also a major binding site for cocaine and other stimulants, the psychoactive effects of which are associated primarily with the inhibition of dopamine reuptake within mesocorticolimbic dopaminergic neurons. We used electron microscopy with an anti-peptide antiserum directed against the N-terminal domain of DAT to determine the subcellular localization of this transporter in the rat ventral tegmental area (VTA), the region that contains the cell bodies and dendrites of these dopaminergic neurons. We show that in the VTA, almost 95% of the DAT immunogold-labeled profiles are neuronal perikarya and dendrites, and the remainder are unmyelinated axons. Within perikarya and large proximal dendrites, almost all of the DAT immunogold particles are associated with intracellular membranes, including saccules of Golgi and cytoplasmic tubulovesicles. In contrast, within medium- to small-diameter dendrites and unmyelinated axons, most of the DAT gold particles are located on plasma membranes. In dually labeled tissue, peroxidase reaction product for the catecholamine-synthesizing enzyme tyrosine hydroxylase is present in DAT-immunoreactive profiles. These findings suggest that intermediate and distal dendrites are both the primary sites of dopamine reuptake and the principal targets of cocaine and related psychostimulants within dopaminergic neurons in the VTA.
Coadministration of κ-opioid receptor agonists (κ-agonists) with cocaine prevents alterations in dialysate dopamine (DA) concentration in the nucleus accumbens (Acb) that occur during abstinence from repeated cocaine treatment. Quantitative microdialysis was used to determine the mechanism producing these effects. Rats were injected with cocaine (20 mg/kg, i.p.), or saline, and the selective κ-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DAext) and extraction fraction (Ed), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increased Ed, whereas repeated U-69593 treatment decreased Ed, relative to controls. Coadministration of both drugs yielded intermediate Ed values not different from controls. In vitro DA uptake assays confirmed that repeated U-69593 treatment produces a doserelated, region-specific decrease in DA uptake and showed that acute U-69593 administration increases DA uptake in a norbinaltorphimine reversible manner. Repeated U-69593 also led to a decrease in [125I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that κ-opioid receptor activation modulates DA uptake in the Acb in a manner opposite to that of cocaine: Repeated U-69593 administration decreases the basal rate of DA uptake, and acute U-69593 administration transiently increases DA uptake. κ-agonist treatment also alters DAT function. The action of κ-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported "cocaine-antagonist" effect of κ-opioid receptor agonists.
The inhibition by cocaine of inward and outward transport of dopamine (DA) at the cloned human norepinephrine transporter (hNET) and the relationship of the inhibitory patterns of cocaine to the conformational requirements of the transporter were investigated. This was done using rotating disk electrode voltammetry in transfected cells. The uphill uptake of external DA, the lack of inhibition by internal substrates on DA uptake, and the accelerated exchange of internal DA by external m-tyramine support a carrier model in which the hNET alternates between outward-facing and inward- facing conformations. Cocaine exhibited competitive inhibition of DA uptake, which was insensitive to intracellular substrates. In contrast, the inhibition by cocaine of the m-tyramine-induced DA efflux appeared noncompetitive relative to intracellular DA, but competitive relative to extracellular m-tyramine. Simultaneous measurement of m-tyramine uptake and accompanying DA efflux at various concentrations of intracellular DA showed that cocaine did not alter the ratio of DA efflux to m-tyramine uptake. Moreover, cocaine displayed similar potency for inhibiting DA uptake and efflux. Additionally, the inhibition profile of cocaine was unrelated to the addition time of cocaine, simultaneously with or earlier than a substrate. All of the findings are consonant with a competitive interaction between cocaine and substrates at the outward-facing conformation of the hNET. This action directly prevents the inward transport of external substrates; thereby inhibiting the outward transport of internal substrates by reducing the availability of the inward-facing conformation. Consequently, the experimental inhibition pattern of cocaine depends on the conformation of the hNET to which the transported substrate is exposed.
Epidemiological studies suggest a link between pesticide exposure and an increased risk of developing Parkinson's disease (PD). Although studies have been unable to clearly identify specific pesticides that contribute to PD, a few human studies have reported higher levels of the organochlorine pesticides dieldrin and DDE (a metabolite of DDT) in post-mortem PD brains. Previously, we found that exposure of mice to dieldrin caused perturbations in the nigrostriatal dopamine system consistent with those seen in PD. Given the concern over the environmental persistence and reintroduction of DDT for the control of malaria-carrying mosquitoes and other pests, we sought to determine whether DDT and its two major metabolites, DDD and DDE, could damage the dopamine system. In vitro analyses in mouse synaptosomes and vesicles demonstrated that DDT and its metabolites inhibit the plasma membrane dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT2). However, exposure of mice to either DDT or DDE failed to show evidence of nigrostriatal damage or behavioral abnormalities in any of the measures examined. Thus, we report that in vitro effects of DDT and its metabolites on components of the dopamine system do not translate into neurotoxicological outcomes in orally exposed mice and DDT appears to have less dopamine toxicity when compared to dieldrin. These data suggest elevated DDE levels in PD patients may represent a measure of general pesticide exposure and that other pesticides may be responsible for the association between pesticide exposure and PD.
Background/Aims:
Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes.
Methods:
The activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR.
Results:
Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na+/H+ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na+/H+ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na+/H+ exchange rate and maintained a lower steady-state pHi, despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7).
Conclusion:
Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pHi of these cells.
by
Ayesha Sultan;
Min Luo;
Qin Yu;
Brigitte Riederer;
Weiliang Xia;
Mingmin Chen;
Simone Lissner;
Johannes E. Gessner;
Mark Donowitz;
Chris Yun;
Hugo deJonge;
Georg Lamprecht;
Ursula Seidler
Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na + /H + exchanger NHE3 is regulated by the Na + /H + Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes ('lipid rafts') were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO 2 /HCO 3 - mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.
With the initial discovery of penicillin and the ensuing mass production of antibiotics in the 1940s, infectious bacteria quickly adapted and developed resistance to the deleterious molecules. In fact, a report published in 1947 found that of 100 staphylococcus infections tested, 38 were classified as highly resistant to penicillin (1). The initial resistance was primarily associated with individual enzymes inactivating specific antibiotics, such as β-lactamases on penicillin. As novel antibiotics were implemented to combat resistant pathogens, selective pressure led to fundamentally new methods of drug resistance. Currently, there are roughly three major mechanisms utilized by bacteria to evade the toxic effects of biocidal agents. These mechanisms include enzymes that modify the drug, alteration of the antibacterial target, and reduced drug uptake due to the presence of efflux pumps or a decrease in porin expression.
Organelle morphology of the endomembrane system is critical for optimal organelle function. ADP ribosylation factors (Arfs), a family of small GTPases, are required for maintaining the structure of the Golgi and endosomes. What determines the discontinuous nature of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as tubulovesicular clusters is unknown. In search of morphological determinants for the ERGIC, we found that a double knockdown of Arf1+Arf4 induced dynamic ERGIC tubules that connect ERGIC clusters, indicating that the tubules mediated lateral intraERGIC traffic. Tubule formation was inhibited by an antagonist of group VI calcium-independent phospholipase A 2 (PLA2G6) and by silencing the A isoform of PLA2G6 (PLA2G6-A). Arf1+Arf4 depletion altered the expression of PLA2G6-A splice variants and relocalized PLA2G6-A from the cytosol to ERGIC clusters and tubules, suggesting that the enzyme became locally active. We show that changes in Arf1 can modulate the activity of PLA2G6-A. We propose that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC in a way that is predicted to impact the rate and possibly the destination of cargos. Our findings have identified key components in the molecular mechanism underlying the regulation of tubules in the ERGIC and uncover tubular carriers as tightly controlled machinery.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.
by
Jeffrey Javidfar;
Ahmed Labib;
Gabrielle Ragazzo;
Ethan Kurtzman;
Maria Callahan;
Silver Heinsar;
Vadim Gudzenko;
Peter Barrett;
Jose Binongo;
Jane Wenjing Wei;
John Fraser;
Jacky Y. Suen;
Gianluigi Li Bassi;
Giles Peek
Previous experience has shown that transporting patients on extracorporeal membrane oxygenation (ECMO) is a safe and effective mode of transferring critically ill patients requiring maximum mechanical ventilator support to a quaternary care center. The coronavirus disease 2019 (COVID-19) pandemic posed new challenges. This is a multicenter, retrospective study of 113 patients with confirmed severe acute respiratory syndrome coronavirus 2, cannulated at an outside hospital and transported on ECMO to an ECMO center. This was performed by a multidisciplinary mobile ECMO team consisting of physicians for cannulation, critical care nurses, and an ECMO specialist or perfusionist, along with a driver or pilot. Teams practised strict airborne contact precautions with eyewear while caring for the patient and were in standard Personal Protective Equipment. The primary mode of transportation was ground. Ten patients were transported by air. The average distance traveled was 40 miles (SD ±56). The average duration of transport was 133 minutes (SD ±92). When stratified by mode of transport, the average distance traveled for ground transports was 36 miles (SD ±52) and duration was 136 minutes (SD ±93). For air, the average distance traveled was 66 miles (SD ±82) and duration was 104 minutes (SD ±70). There were no instances of transport-related adverse events including pump failures, cannulation complications at outside hospital, or accidental decannulations or dislodgements in transit. There were no instances of the transport team members contracting COVID-19 infection within 21 days after transport. By adhering to best practices and ACE precautions, patients with COVID-19 can be safely cannulated at an outside hospital and transported to a quaternary care center without increased risk to the transport team.