Releaser vs Reuptake Inhibitor: question on drug competition binding assay

[DotChem]

In competition binding assays to determine drug activity, one measures the displacement of a standard compound by the drug to be tested from the receptor. For example, to test if the drug is a dopamine transporter ligand, one measures the displacement of the standard ligand WIN 35428 from DAT preparations. You first incubate the brain preparations containing DAT with the standard WIN ligand for a certain time (say 120 mins) and then you add your test drug and measure how much radioactivity is released ie how much WIN has been displaced from the transporter. By varying the concentration of your test compound you then determine its Ki.
Now my question is: In the specific case of transporters done this way, does that not make the test drug a releaser rather than a reuptake inhibitor. First letting the standard drug binds to the receptor by incubating for 120mins and then adding the drug to be tested. So assuming WIN 35428 binds to the same site as the Dopamine substrate, would that not make compounds tested in this way releasers rather than reputake inhibitors? Because in the case of transporters, this makes a huge difference in terms of the resulting psychopharmacological response whether the drug is acting by blocking the binding of the neurotrasmitter to its transporter in the frirst place or releasing already bound neurotransmitter: your take on this.

 

[Coolwhip]

I'm not sure I follow, but I don't think you could tell which it was at that point. But why would you think its a releaser just because it displaces WIN-35428(which is an inhibitor)?

 

[serotonin2A]

I don't really understand your criticism. The assays are usually performed using homogenized membrane so DAT is not functional, ie there is no transport or endogenous transmitter so no difference between releasers and re-uptake inhibitors.

But even in your example, the compounds tested wouldn't necessarily act as releasers. Releasers and re-uptake inhibitors differ in terms of how they specifically interact with DAT, and the assay conditions wouldn't change the nature of the interaction.

 

[DotChem]

I guess my question is: how does one differentiate between a releaser and a reuptake inhibitor since the pharmacological response between the 2 is quite different? I mean one induces the release of previously taken up neurotransmitter by synaptosomes and the other compete to prevent their taken up in the first place.

 

[serotonin2A]

I guess my question is: how does one differentiate between a releaser and a reuptake inhibitor since the pharmacological response between the 2 is quite different? I mean one induces the release of previously taken up neurotransmitter by synaptosomes and the other compete to prevent their taken up in the first place.

It is not possible to differentiate between uptake inhibitors and releasers based on competitive binding assays. At the very least, you also have to measure whether a ligand also has substrate activity.

 

[aced126]

When DAT moves a substrate into the cytosol, 2 Na+ are transported as well. Thus releasers can be distinguished from reuptake inhibitors by measuring a change in membrane potential.

 

[serotonin2A]

When DAT moves a substrate into the cytosol, 2 Na+ are transported as well. Thus releasers can be distinguished from reuptake inhibitors by measuring a change in membrane potential.

The question was specifically about competitive binding assays, so that's what I was referring to. They are performed on membrane fractions, which have no transmembrane potential and hence DAT cannot transport substrates.

 

[Limpet_Chicken]

Tramsporter phosphorylation via kinases is important too.

 

[DotChem]

Thanks guys.. but most papers use competition binding assay to determine monoamine release and draw conclusion on the effect of the drug: competition for binding to the transporter that is. here is an example from this paper measuring mephedrone, cathinone and Meth Monoamine Release. It seems to be standard assay everybody follows. here is the in vitro monoamine release procedure part: full paper here: "The Designer Methcathinone Analogs, Mephedrone and Methylone, are Substrates for Monoamine Transporters in Brain Tissue https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306880/ "

In our initial investigations, we tested the ability of mephedrone, methylone, MDMA, and methamphetamine to evoke the release of radiolabeled substrates in rat brain synaptosomes using modifications of published methods (Rothman et al, 2001). Briefly, in vitro release assays were conducted using [3H]MPP+ as the radiolabeled substrate for norepinephrine transporters (NET) and dopamine transporters (DAT), while using [3H]5-HT as the radiolabeled substrate for 5-HT transporters (SERT). Whole brain minus cerebellum (for NET and SERT assays) or striatum (for DAT assays) was homogenized in ice-cold 10% sucrose containing 1 mM reserpine. For the NET release assays, 100nM GBR12935 and citalopram were added to the sucrose solution to block [3H]MPP+ uptake into DA and 5-HT terminals. For DAT release assays, 100nM desipramine and citalopram were added to block [3H]MPP+ uptake into NE and 5-HT terminals. For SERT release assays, 100nM nomifensine and GBR12935 were added to block uptake of [3H]5-HT into NE and DA terminals. After 12 strokes with a Potter–Elvehjem homogenizer, homogenates were centrifuged at 1000g for 10 min at 4 1C, and the supernatants (ie, synaptosomes) were retained on ice. Synaptosomes were incubated to steady state in a polypropylene beaker, with stirring at 25 1C, in Krebs-phosphate buffer (pH 7.4), which contained 1 mM reserpine and either 5 nM [3H]MPP+ or 5nM[3H]5-HT. To commence the assay, 850 ml of preloaded synaptosomes were added to polystyrene test tubes or 96-well plates that contained 150 ml test drug in uptake buffer plus 1 mg/ml bovine serum albumin. After 30 min ([3H]MPP+ assays) or 5 min ([3H]5-HT), the release reaction was terminated by dilution with 4ml wash buffer (10mM Tris-HCl pH 7.4 containing 0.9% NaCl at 25 1C) followed by rapid vacuum filtration over Whatman GF/B filters using a Brandel cell harvester (Brandel, Gaithersburg, MD). Filters were rinsed twice with 4ml wash buffer and dried under vacuum. The retained tritium was counted by a Trilux liquid scintillation counter (Micromedic Systems, Philadelphia, PA) at 40% efficiency after an overnight extraction in 0.6 ml scintillation cocktail...

As I said, this asays is basically measuring drug induced release of radioactive ligands PREVIOUSLY taken up by transporter preparations by competitive displacement (MPP+ for DAT and NET and tritiated 5HT for SERT). I think I am missing something here (as a synthetic chemist by training not a neuropharmacologist.. thx guys and y'all have a good weekend

 

[serotonin2A]

The study you just cited is not a competitive binding assay. The example you cited in your first post used tritiated WIN 35428 to label DAT, and then measured displacement by other ligands. That is a competitive binding assay. The methodology used in the last study is completely different