Differences between DA releasers and reuptake inhibitors

[aced126]

I am finding it difficult to understand the following article (it analyses the pharmacophores for releasing potency and reuptake inhibition of DA):

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867964/pdf/cn4001236.pdf

What does it mean by an inward or outward current? And why would reuptake inhibitors and releasers display these different current patterns. What other experimental techniques are available to determine if a compound acts as a releaser or reuptake inhibitor? The study states specifically "an outward current represents the block of an endogenous leak current". Why is this so?

I am assuming the inward current spike when DA itself is applied is due to the DA triggering an action potential at a postsynaptic membrane. Why is the secondary inward spike (when DA is administered very shortly after a reuptake inhibitor is administered) of a much smaller magnitude than the initial inward spike?

 

[serotonin2A]

What they mean by "inward current" is that ions are flowing from the outside to the inside of the cell. It is simply a way to specify the direction of net current flow relative to the cell membrane.

Substrate transport by DAT occurs because there is a transmembrane gradient of sodium ions. The movement of sodium into cells is energetically favorable and is the driving force behind DA transport by DAT. Sodium first binds to the extracellular part of DAT, then dopamine binds, and then a conformational change occurs that moves dopamine and the sodium ion inside the cell. So the transport of dopamine into the cell also produces an inward current composed of sodium ions.


The inward current triggered by DA occurs because the dopamine is taken up into the oocyte, which causes sodium ions to be transported into the cell. Dopamine is also positively charged at physiological pH and so transport of DA into the cell also contributes to the inward current.

The second spike of inward current is much smaller than the first because MDPV (or analog) blocks the uptake of DA, hence the amount of sodium/dopamine transport is reduced in the presence of MDPV.


These types of experiments can be used to distinguish releasing agents vs. reuptake inhibitors. DA releasers act as DAT substrates and induce an inward current. Conversely, uptake inhibitors block the inward current elicited by DA and produce a small outward current, which represents the inhibition of a small leak current that occurs spontaneously.

 

[aced126]

What they mean by "inward current" is that ions are flowing from the outside to the inside of the cell. It is simply a way to specify the direction of net current flow relative to the cell membrane.

Substrate transport by DAT occurs because there is a transmembrane gradient of sodium ions. The movement of sodium into cells is energetically favorable and is the driving force behind DA transport by DAT. Sodium first binds to the extracellular part of DAT, then dopamine binds, and then a conformational change occurs that moves dopamine and the sodium ion inside the cell. So the transport of dopamine into the cell also produces an inward current composed of sodium ions.


The inward current triggered by DA occurs because the dopamine is taken up into the oocyte, which causes sodium ions to be transported into the cell. Dopamine is also positively charged at physiological pH and so transport of DA into the cell also contributes to the inward current.

The second spike of inward current is much smaller than the first because MDPV (or analog) blocks the uptake of DA, hence the amount of sodium/dopamine transport is reduced in the presence of MDPV.


These types of experiments can be used to distinguish releasing agents vs. reuptake inhibitors. DA releasers act as DAT substrates and induce an inward current. Conversely, uptake inhibitors block the inward current elicited by DA and produce a small outward current, which represents the inhibition of a small leak current that occurs spontaneously.

Thanks, that clears a lot up. I'll probably ask a few more questions when I have more time to look into this further.

 

[aced126]

Why do reuptake inhibitors cause an outward current? My understanding is that the extracellular DA and Na+ ions are blocked from entering. But why is there any extracellular DA anyway? The graph stabilises (to the pre-DA current) after the initial DA peak, shortly before MDPV administration which I think would imply all the extracellular DA has been taken up.

What would the plot look like if amphetamine is administered for 1 minute instead of MDPV? I'd imagine an initial outward current but what happens when amphetamine causes phosphorylation of DAT? How long does this take to happen anyway? And once it happens, does DAT transport a DA molecule and a Na+ ion from inside the oocyte to the extracellular environment?

I guess what I'm trying to say is how one could distinguish from a DAT substrate (like reserpine) vs a releaser, which is a substrate in itself but also other things?

 

[serotonin2A]

Why do reuptake inhibitors cause an outward current? My understanding is that the extracellular DA and Na+ ions are blocked from entering. But why is there any extracellular DA anyway? The graph stabilises (to the pre-DA current) after the initial DA peak, shortly before MDPV administration which I think would imply all the extracellular DA has been taken up.

The outward current has nothing to do with extracellular DA. At baseline there is a slight inward current due to ions leaking through DAT. Reuptake inhibitors block the inward current, which effectively shifts the baseline. You can see that in the figure from the paper.

What would the plot look like if amphetamine is administered for 1 minute instead of MDPV? I'd imagine an initial outward current but what happens when amphetamine causes phosphorylation of DAT? How long does this take to happen anyway? And once it happens, does DAT transport a DA molecule and a Na+ ion from inside the oocyte to the extracellular environment?

First of all, the oocytes are not terminals -- there is no specific mechanism to phosphorylate DAT, no TAAR1, no vesicles with VMAT2. If they had applied amphetamine to the oocytes then it would have acted like any other substrate.

EDIT: Here you can see what amphetamine does to oocytes expressing DAT. Intracellular amphetamine strangely does induce a persistent leak current, but that effect has nothing to do with DAT phosphorylation or dopamine transport -- the current is dependent on extracellular sodium and occurs in the absence of dopamine. It appears that in the presence of intracellular amphetamine DAT can adopt a conformation that is permeable to sodium ions (ie, it acts like an ion channel).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423253/

I guess what I'm trying to say is how one could distinguish from a DAT substrate (like reserpine) vs a releaser, which is a substrate in itself but also other things?

These experiments are not designed to distinguish substrates vs releasers. You would have to use a different technique.

 

[Nagelfar]

These types of experiments can be used to distinguish releasing agents vs. reuptake inhibitors. DA releasers act as DAT substrates and induce an inward current. Conversely, uptake inhibitors block the inward current elicited by DA and produce a small outward current, which represents the inhibition of a small leak current that occurs spontaneously.

Might this perhaps, explain why rate of firing is increased with DRIs and slowed with DRAs? I figured it had something to do with potential endogenous ligands and a natural evolution for reuptake-pump ligands to be considered to block both directions as per the "atypical inhibitors" like GBR compounds and benztropines, and that methylphenidate & cocaine are anomalies in the case of doing just one without inhibiting spontaneous release and the rate of fire is not augmented as the body's attempt at homeostasis. However some other mechanism for this possibility would be interesting, I don't know if inward or outward current could convey to something like that by whatever means but it'd be interesting if so.

 

[serotonin2A]

Might this perhaps, explain why rate of firing is increased with DRIs and slowed with DRAs? I figured it had something to do with potential endogenous ligands and a natural evolution for reuptake-pump ligands to be considered to block both directions as per the "atypical inhibitors" like GBR compounds and benztropines, and that methylphenidate & cocaine are anomalies in the case of doing just one without inhibiting spontaneous release and the rate of fire is not augmented as the body's attempt at homeostasis. However some other mechanism for this possibility would be interesting, I don't know if inward or outward current could convey to something like that by whatever means but it'd be interesting if so.

The effect could potentially increase the excitability of dopaminergic neurons. DAT is primarily expressed on terminals, where an increase in depolarizing Na+ current would open Ca2+ channels and increase spontaneous vesicular docking and transmitter release. So in the terminals, DAT-mediated depolarization could potentially increase DA release but it wouldn't change the firing rate. However, DAT is also expressed on dendrites of dopaminergic neurons. If DAT depolarizes the dendrites then the firing rate may increase.

 

[DotChem]

AMPH-like releasers induce depolarization of the neuron membrane while the reuptake inhibitors such as MDPV/cocaine induce hyper-polarization instead. That's probably the best way to distinguish between the 2. Though not quite sure about the mechanism, it seems releasers also induce DAT reversal translocation from pre-synaptic storage vesicles back to the membrane. What is not clear to me if Na+ is also co-transported back out to the synaptic cleft along with dopamine(inhibitor) sent to storage. In that case the current will be reversed. Or the reverse translocation of the DAT protein is that of the unbound DAT transporter following storage of dopamine(inhibitor) in storage vesicles.

In other word, while both compete with the dopamine substrate for binding to the transporter, the releasers such as AMPH recycle Na+bound transporter (ie DAT*Na+) while the reuptake inhibitors such as MDPV do notthey basically block the reuptake and the translocation of the dopamine(inhibitor)-bound transporter in the first place. In other word once the dopamine substrate is displaced by competing inhibitors for binding to DAT, releasers AMPH-like are transported just like the substrate to storage vesicles and then back out while reuptake inhibitors are not. Thus the releasers inducing depolarisation and current reversal and the reuptake inhibitors hyper-polarization?..

https://en.wikipedia.org/wiki/Transporter_reversal

Most neurotransmitter transporters are involved in the reuptake of neurotransmitters back into the cell which released them; during neurotransmitter reuptake, these transporter will move specific types of neurotransmitters from the extracellular space into the cytoplasm of a neuron or glial cell. When these transporters operate in reverse, they produce neurotransmitter efflux (i.e., the movement neurotransmitters from the cytoplasm into the extracellular space). In neurons, transporter reversal facilitates the release of neurotransmitters into the synaptic cleft, which subsequently increases the binding of these neurotransmitters at their associated neurotransmitter receptors. For example, many monoamine releasing agents produce monoamine efflux (i.e., the release of monoamine neurotransmitters from neurons into the synaptic cleft) by triggering reverse transport at vesicular monoamine transporters (specifically VMAT1 and VMAT2) and other monoamine transporters that are located along the plasma membrane of neurons (specifically, DAT, NET, and SERT).

Amphetamine and similar "releasing agents" are able to reverse the transport direction of monoamine transporters through the activation of an intracellular receptor....etc

 

[serotonin2A]

AMPH-like releasers induce depolarization of the neuron membrane while the reuptake inhibitors such as MDPV/cocaine induce hyper-polarization instead. That's probably the best way to distinguish between the 2. Though not quite sure about the mechanism, it seems releasers also induce DAT reversal translocation from pre-synaptic storage vesicles back to the membrane.

In terms of cocaine-induced hyperpolarization, are you referring to the effect in this paper?

http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.1990.tb12998.x/pdf

The hyperpolarization of dopaminergic neurons by cocaine is thought to occur due to activation of D2 autoreceptors, which is an indirect effect.

 

[DotChem]

No but the first paper mentioned on this thread + this one:

"Mephedrone and Methylenedioxypyrovalerone (MDPV), Major Constituents of Bath Salts, Produce Opposite Effects at the
Human Dopamine Transporter*" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3881434/

Both cocaine and MDPV induce hyperpolarization of oocytes expressing human DAT, while AMPHs like methamphetamine and methcathinone induce depolarization of the neuron membrane:

At −60 mV, 10 μM S-methamphetamine and S-methcathinone (Figure 1) produced an
inward (depolarizing) current through hDAT; mephedrone produced similar effects under
the same conditions (representative tracings are shown in Figure 2). All three agents
generated a sustained leak (‘shelf’) current that persisted long after the drug was removed......

...In contrast to methamphetamine, methcathinone, or mephedrone, MDPV (10 μM) produced
an outward, cocaine-like, hyperpolarizing current at −60 mV which, in actuality, shows
inhibition of the endogenous leak current of hDAT (Sonders et al, 1997). That is, both
MDPV and cocaine (examined at six different concentrations ranging from 0.01 to 30 μM;
Figure 4) produced similar inhibitory effects, with MDPV producing a greater effect
(remaining % of dopamine pre-pulse = 32.9 ± 1.9% and 24.6 ± 0.5%, respectively, n=5;
two-tailed T test: p<0.01, t = 4.23, df = 8), and were of similar potency (EC50 = 0.33 ± 0.07
μM and 0.30 ± 0.04 μM, respectively, n=5) (p>0.05, t = 0.36, df = 8) in the inhibition of
hDAT endogenous leak..

...Thus, MDPV produces a hyperpolarizing current consistent with hDAT blockade..

So I guess that is in addition to cocaine inducing hyperpolarization via agonism at D2 autoreceptor mechanism.

 

[serotonin2A]

The effects you are talking about were seen in oocytes engineered to express DAT, and are not observed when recordings are made from dopaminergic cells. That doesn't mean that those effects are absent in neurons, but it isn't accurate to say that neuronal membranes are hyperpolarized or depolarized by a mechanism unless the effect can actually be observed in recordings. One reason they use ooctyes to study the function of DAT is that it makes it possible to detect effects that cannot be observed in neurons.

You can see the effects in neurons here:

http://www.ncbi.nlm.nih.gov/pubmed/2361170

The outward current is blocked by sulpiride, a D2/3 receptor antagonist, meaning it is likely mediated by autoreceptor activation.

 

[Nagelfar]

The effect could potentially increase the excitability of dopaminergic neurons. DAT is primarily expressed on terminals, where an increase in depolarizing Na+ current would open Ca2+ channels and increase spontaneous vesicular docking and transmitter release. So in the terminals, DAT-mediated depolarization could potentially increase DA release but it wouldn't change the firing rate. However, DAT is also expressed on dendrites of dopaminergic neurons. If DAT depolarizes the dendrites then the firing rate may increase.

Firing rate may increase with de-polarization, meaning releasers/substrates like amph? or would the spontaneous vesicular docking caused via increase in depolarizing sodium channels, being by way of on terminals (external to the cell membrane a la DRIs?) and the Ca2+ channels help explain the 'inverse-agonist' hypothesis of making a downstream effect usually only accounted for in releasers?

 

[serotonin2A]

Firing rate may increase with de-polarization, meaning releasers/substrates like amph? or would the spontaneous vesicular docking caused via increase in depolarizing sodium channels, being by way of on terminals (external to the cell membrane a la DRIs?) and the Ca2+ channels help explain the 'inverse-agonist' hypothesis of making a downstream effect usually only accounted for in releasers?

Sorry, I'm having trouble following your question. Monoamine releasers act by several different potential mechanisms that are not mutually exclusive. So amphetamine may work by reversing transport and also by switching DAT into a channel mode. DAT-induced depolarization of terminals is an additional mechanism through which amphetamine can increase monoamine release. Depolarization of dendrites could also potentially increase monoamine release, but that effect would be offset by autoreceptor activation.

 

[Nagelfar]

Sorry, I'm having trouble following your question. Monoamine releasers act by several different potential mechanisms that are not mutually exclusive. So amphetamine may work by reversing transport and also by switching DAT into a channel mode. DAT-induced depolarization of terminals is an additional mechanism through which amphetamine can increase monoamine release. Depolarization of dendrites could also potentially increase monoamine release, but that effect would be offset by autoreceptor activation.

My question had to do with DRAs as contrasting DRIs and what ways DRIs act differently that may be potentiating, as its well known that DRAs have additional efficacious-to-stimulation modes of actions over DRIs, I'm looking for the opposite from some leads I've read on in this subject-direction

 

[DotChem]

The outward current is blocked by sulpiride, a D2/3 receptor antagonist, meaning it is likely mediated by autoreceptor activation.

I see. If the D2/3 antagonist sulpiride blocks the hyperpolarization effect of cocaine, that would strongly points to activation at D2 autoreceptors as the mechanism underlying cocaine effect, isn't it? However, D2/3 activation do not fully account for the response (current) elicited by cocaine in midbrain dopaminergic neurons. see :
"Dopamine transporter-mediated conductances increase excitability of midbrain dopamine neurons." https://www.ncbi.nlm.nih.gov/pubmed/12352983

Uptake by Na(+)/Cl(-)-dependent neurotransmitter transporters is the principal mechanism by which extracellular biogenic amine concentrations are regulated. In addition to uptake, the cloned transporter proteins also elicit ion channel-like currents, but the physiological consequences of these currents are unknown. Here, whole-cell patch clamp and perforated-patch recordings show that substrates of the dopamine transporter (DAT), such as dopamine (DA) and amphetamine, increase the firing activity of rat DA neurons in culture. We found that these substrates elicit inward currents that are Na(+)-dependent and blocked by cocaine. These currents are primarily comprised of anions and result in an excitatory response in DA neurons at lower DA concentrations than are required for D2 autoreceptor activation. Thus, in addition to clearing extracellular DA, our results suggest that the currents associated with DAT modulate excitability and may regulate release of neurotransmitter from midbrain DA neurons.

Now as to MDPV which was actually the subject of the paper cited at the beginning of this thread (how to differentiate cocaine-like DRI and AMPH-like DRA?) the question is: could it also be eliciting the hyperpolarization effect observed with cocaine by activation of D2/D3 or by virtue of DAT modulation? Actually more strongly that cocaine. The aim of the paper as I've understood it is to develop an assay that would differentiate the AMPH-like stimulants from the cocaine-like. Oocytes expressing human DAT provide a pretty model imho. But it would be more relevant to investigate the effect of MDPV on dopaminergic neurons as in the paper you've cited and also its effect on D2/D3 autoreceptors. .. (thx for your input: As always I learn a lot on this forum...

 

[serotonin2A]

You have to remember that there is a difference between what ion channels/proteins in neuronal soma and dendrites do locally and what actually happens at the whole cell level. Patch clamping is a great way to measure what individual channels or proteins can do, but often that doesn't actually translate to changes in cell activity. Same with cell culture experiments. Slice recordings are much more definitive ways to understand what a drug is likely to do in the brain. There are certainly situations when slice recordings miss effects that occur in vivo, but patch clamp recordings from cultured cells have even less physiological relevance than slice recordings. The effects they are detecting with cocaine in culture probably also occur in the brain but the slice work suggests that the effects may not be strong enough to be physiologically relevant at the whole cell level.

In other words, it is possible to study drug effects that lack relevance to the in vivo situation.

I guess this is getting away from the question of how to differentiate DRA from DRIs. But the discussion took a turn toward effects on DAergic neuron activity, and in that case you have to be aware of the degree to which the model system is relevant to the situation in vivo.

 

[aced126]

To what extent can the modes of actions of DRAs and DRIs be attributed to factors other than the well described ones that do not actually require a neuron to study, but an oocyte in this case (reuptake inhibition, DA release etc). For example, could altered expression of genes play a role (I'm guessing not as this would take too much time to be of acute observable effect).

I don't mind at all if the topic of discussion moves away from the original topic, as long as it stimulates discussion.

Also wanted to thank all the regular posters in NSPD, especially serotonin2A, for all the precise and insightful input provided not only now but to every single question I ask and other peoples' questions as well. Like DotChem, I have gained directly and indirectly enormous knowledge and understanding in this forum.

 

[Nagelfar]

Like DotChem, I have gained directly and indirectly enormous knowledge and understanding in this forum.

if we're lauding praise, then count me among those, in the nearly decade I've been here I've become a fool bumbling in the dark to someone who has had the (albeit fleetingly) ability to ask someone with multiple PhDs specializing in molecular physic-type theory a thought provoking question (as one would guess to be in their opinion rather than my own as to what that amounts to)

The amazing part, is, the original considerations for which I had joined, have only broadened the mystery inherent in those first questions, the more we know, the more we understand that we do not; or as Isaac Newton said: "the larger the continent of knowledge, the longer the shore-line of the unknown"