Ion Dependence of Carrier-Mediated Release in Dopamine or Norepinephrine Transporter-Transfected Cells Questions the Hypothesis of Facilitated Exchange Diffusion

1999 ◽  
Vol 56 (5) ◽  
pp. 1047-1054 ◽  
Author(s):  
Christian Pifl ◽  
Ernst A. Singer
Keyword(s):  
Norepinephrine Transporter ◽  
Transfected Cells ◽  
Exchange Diffusion

Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.

1995 ◽  
Vol 198 (10) ◽  
pp. 2197-2212 ◽  
Author(s):  
A Galli ◽  
L J DeFelice ◽  
B J Duke ◽  
K R Moore ◽  
R D Blakely
Keyword(s):  
Radioligand Binding ◽  
Cell Voltage ◽  
Norepinephrine Transporter ◽  
Charge Movement ◽  
Coupled Transport ◽  
Patch Clamp Recording ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Transfected Cells ◽  
293 Cells

Transport of norepinephrine (NE+) by cocaine- and antidepressant-sensitive transporters in presynaptic terminals is predicted to involve the cotransport of Na+ and Cl-, resulting in a net movement of charge per transport cycle. To explore the relationship between catecholamine transport and ion permeation through the NE transporter, we established a human norepinephrine transporter (hNET) cell line suitable for biochemical analysis and patch-clamp recording. Stable transfection of hNET cDNA into HEK-293 (human embryonic kidney) cells results in lines exhibiting (1) a high number of transporter copies per cell (10(6)), as detected by radioligand binding and hNET-specific antibodies, (2) high-affinity, Na(+)-dependent transport of NE, and (3) inhibitor sensitivities similar to those of native membranes. Whole-cell voltage-clamp of hNET-293 cells reveals NE-induced, Na(+)-dependent currents blocked by antidepressants and cocaine that are absent in parental cells. In addition to NE-dependent currents, transfected cells posses an NE-independent mode of charge movement mediated by hNET. hNET antagonists without effect in non-transfected cells abolish both NE-dependent and NE-independent modes of charge movement in transfected cells. The magnitude of NE-dependent currents in these cells exceeds the expectations of simple carrier models using previous estimates of transport rates. To explain our observations, we propose that hNETs function as ion-gated ligand channels with an indefinite stoichiometry relating ion flux to NE transport. In this view, external Na+ and NE bind to the transporter with finite affinities in a cooperative fashion. However, coupled transport may not predict the magnitude or the kinetics of the total current through the transporter. We propose instead that Na+ gates NE transport and also the parallel inward flux of an indeterminate number of ions through a channel-like pore.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals A proof of principle study of atomoxetine for the prevention of vasovagal syncope: the Prevention of Syncope Trial VI

EP Europace ◽  
2019 ◽  
Vol 21 (11) ◽  
pp. 1733-1741 ◽  
Author(s):  
Robert S Sheldon ◽  
Lucy Lei ◽  
Juan C Guzman ◽  
Teresa Kus ◽  
Felix A Ayala-Paredes ◽  
...  
Keyword(s):  
Cardiac Index ◽  
Stroke Volume ◽  
Vasovagal Syncope ◽  
Resistance Index ◽  
Stroke Volume Index ◽  
Study Drug ◽  
Norepinephrine Transporter ◽  
Volume Index ◽  
Head Up Tilt ◽  
Invasive Techniques

Abstract Aims There are few effective therapies for vasovagal syncope (VVS). Pharmacological norepinephrine transporter (NET) inhibition increases sympathetic tone and decreases tilt-induced syncope in healthy subjects. Atomoxetine is a potent and highly selective NET inhibitor. We tested the hypothesis that atomoxetine prevents tilt-induced syncope. Methods and results Vasovagal syncope patients were given two doses of study drug [randomized to atomoxetine 40 mg (n = 27) or matched placebo (n = 29)] 12 h apart, followed by a 60-min drug-free head-up tilt table test. Beat-to-beat heart rate (HR), blood pressure (BP), and cardiac haemodynamics were recorded using non-invasive techniques and stroke volume modelling. Patients were 35 ± 14 years (73% female) with medians of 12 lifetime and 3 prior year faints. Fewer subjects fainted with atomoxetine than with placebo [10/29 vs. 19/27; P = 0.003; risk ratio 0.49 (confidence interval 0.28–0.86)], but equal numbers of patients developed presyncope or syncope (23/29 vs. 21/27). Of patients who developed only presyncope, 87% (13/15) had received atomoxetine. Patients with syncope had lower nadir mean arterial pressure than subjects with only presyncope (39 ± 18 vs. 69 ± 18 mmHg, P < 0.0001), and this was due to lower trough HRs in subjects with syncope (67 ± 30 vs. 103 ± 32 b.p.m., P = 0.006) and insignificantly lower cardiac index (2.20 ± 1.36 vs. 2.84 ± 1.05 L/min/m2, P = 0.075). There were no significant differences in stroke volume index (32 ± 6 vs. 35 ± 5 mL/m2, P = 0.29) or systemic vascular resistance index (2156 ± 602 vs. 1790 ± 793 dynes*s/cm5*m2, P = 0.72). Conclusion Norepinephrine transporter inhibition significantly decreased the risk of tilt-induced syncope in VVS subjects, mainly by blunting reflex bradycardia, thereby preventing final falls in cardiac index and BP.

scholarly journals Reduced Endothelin-2 and Hypoxic Signaling Pathways in Granulosa-Lutein Cells of PCOS Women

2021 ◽  
Vol 22 (15) ◽  
pp. 8216
Author(s):  
Magdalena Szymanska ◽  
Ketan Shrestha ◽  
Eliezer Girsh ◽  
Avi Harlev ◽  
Iris Eisenberg ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Target Genes ◽  
Hypoxic Response ◽  
Transfected Cells ◽  
Healthy Women ◽  
Periovulatory Follicles

Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210—designated hypoxiamiR—and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.

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