I was trying to point out that there is a flaw in your interpretation here. Your logical argument is:
* Amphetamine signals through the cAMP/PKA pathway by activating TAAR1
* And amphetamine stimulates cAMP/PKA, which inhibits RhoA-induced DAT internalization
* Therefore, amphetamine must inhibit RhoA by activating TAAR1
This argument is not valid -- the only way to know if TAAR1 is involved in the effect of amphetamine on RhoA is to actually test the involvement of TAAR1 in an experiment. Often, biochemical events that seem like they should be linked are not linked. Intracellular signaling cascades are segregated into very discrete cellular subdomains, to the point where upstream and downstream effectors are often physically linked by scaffolding proteins. In many cases there is no communication between upstream and downstream signaling partners unless they are actually physically linked. Amphetamine may be able to activate PKA via TAAR1, but that doesn't mean that TAAR1 activates the same PKA subpopulation that regulates DAT internalization in the study you cited. Amphetamine can influence cAMP/PKA through multiple non-TAAR1 mechanisms, for example via indirect effects on dopamine receptors, so there is no way to specifically link the findings in the study to TAAR1 without further hypothesis testing.
This is a fair point. It has not been demonstrated experimentally. It is the most parsimonious explanation however, which is why I'm inclined to believe that this is the case.
I would argue that the effects of amphetamine on vesicles and on DAT are not independent or discrete events, but rather linked processes. You acknowledge that amphetamine releases dopamine via DAT, but where do you think that the released dopamine comes from? Studies have shown that the dopamine released by amphetamine comes from the vesicular pool. Depleting the vesicular pool of dopamine blocks amphetamine-induced dopamine release. Additionally, the action of amphetamine on vesicles is sufficient to induce some extracellular DA efflux because the leakage results in a proportional increase in cytosolic dopamine, which is sufficient to drive some reverse transport. However, amphetamine also has several direct and indirect effects on DAT function that markedly augment the reverse transport of dopamine after it leaks out of vesicles. In effect, studies examining the effect of amphetamine on DAT function are not really studying the phenomenon that causes the dopamine efflux, but rather are studying the phenomenon that regulates the magnitude of dopamine release.
Think about the following situation: you cause a large grease fire and your kitchen, so you open a window to let out the smoke. Someone smells the smoke outside your window and calls the fire department. Was the smoke outside of your house caused by the grease fire, or was it caused by the window being opened? I think most people would argue the smoke was caused by the fire and not by opening the window.
So my point is that when you make statements like "TAAR1-mediated signaling cascades account for roughly 50% of amphetamine-induced DA efflux (via PKC)" are factually incorrect because they attempt to link DA release to a facilitory or regulatory mechanism (amphetamine effects on DAT) rather than to the actual driving force behind amphetamine-induced dopamine release (effects on vesicular dopamine storage).
Obviously, the dopamine which is released through DAT is derived from cytosolic dopamine which is released from VMAT2. You've seen the diagram that I drew, so you should know that I know that. I seldom read individual primary studies because they're not of any use for citing on Wikipedia and don't draw connections among related research. I read reviews which do draw those connections.
This review on kinase-dependent regulation of monoamine transporters draws some of those connections between amphetamine and PKC/CAMKII/PKA/RhoA-dependent phosphorylation of DAT. It also states "Consistent with this idea, PKCb knockout mice demonstrate reduced, though notably not completely eliminated, AMPH-evoked DA efflux (Chen et al., 2009). Similar results were recently observed by the Gnegy group, who found that perfusion of PKCb inhibitors into the nucleus accumbens of rats reduced AMPHevoked DA efflux by approximately 50% (Zestos et al., 2016)." This is one of 2 or 3 sources which I was paraphrasing when I said that amphetamine-induced phosphorylation of DAT via PKC accounts for roughly half of total DA efflux. I'll look for the source I'm paraphrasing about CAMKII-mediated DAT phosphorylation accounting for another 50% if you care to see a source for that too (I can't remember which one stated this off the top of my head).
As for VMAT2 efflux "driving" dopamine release by amphetamine, I don't see what evidence you're basing this on to assert this. Amphetamine induces efflux through DAT via signaling cascades that involve kinase-dependent transporter phosphorylation. Dumping dopamine into the cytosol doesn't cause transporter phosphorylation unless dopamine signals through an intracellular biomolecular target that induces transporter phosphorylation via a protein kinase. This is because DA itself doesn't donate a phosphate group to the protein. DA does signal through TAAR1, so I suppose that you could assert that dumping DA into the cytosol would induce efflux through DAT via that mechanism, but it's a fairly tenuous argument that it also does so by some other unknown means without evidence to support that claim.
Sure, constitutive knockouts can produce compensatory regulations that modify drug responses. But you are arguing that "TAAR1-mediated signaling cascades account for roughly 50% of amphetamine-induced DA efflux (via PKC) and all reuptake inhibition after 30 minutes post-exposure", which would undoubtedly make TAAR1 activation the primary mechanism for amphetamine-induced dopamine release. It isn't a good sign for a hypothesized receptor-based mechanism if knocking out the receptor does not cause some attenuation of the response. There certainly could be alternative reasons for such an outcome -- such as the D2 explaination you proposed -- but the data from TAAR1 knockouts will represent a negative finding until experimental evidence emerges showing that such knockout data should be dismissed.
I said the data was not controlled, not that it should be dismissed. The implication was that it should be interpreted in context with findings of receptor function via other methods and not in isolation
because it is not a controlled system. As an example,
this review states "Alternatively, there may be some adaptive compensatory changes in the dopamine system of the TAAR1 knockout mice. This concept is supported by the observation that these mice have a 262% increase in the proportion of striatal high-affinity D2 receptors (Wolinsky et al., 2006)." I've read a newer review that elaborates further on this but don't have it on my laptop at the moment. I'll upload a link a little later.
I never stated that you argued that all monoamine releasers are TAAR1 agonists. What I actually wrote is the following: "You are specifically arguing that monoamine releasers can be defined as drugs that activate TAAR1 and are transported into monoamine neurons."
A definition is a logical equivalence. So, if you say "A" is defined by "B", where A is a term and B is a statement, then mathematically, set A and set B have identical elements, meaning A is a subset and superset of B. In this context, you defined "A" as "monoamine releasers" and "B" as "drugs that activate TAAR1 and are transported into monoamine neurons", but A is not defined by B as I've argued above (where I've argued that B is a subset of A, but A is not a subset of B - it's a strict superset of B since it contains elements [i.e., drugs] which are not contained in set B). If this wasn't what you meant, you should probably clarify.
I never claimed that you think that TAAR1 is the singular target of amphetamine. What I actually said is that you seem to believe that TAAR1 is the most important target of amphetamine. If you think that TAAR1 is responsible for 50% of the dopamine release produced by amphetamine, then that would make TAAR1 by far one of the most important mediators of amphetamine-induced dopamine release. I don't think the data supports that specific conclusion.
Hmm. Not yet perhaps. The data supports the statement that PKC is responsible for ~50% of DA efflux. The data also supports the statement that TAAR1-mediated PKA and PKC signaling cascades are involved in amphetamine-induced DA reuptake inhibition and efflux respectively. Linking those two statements is simply the most parsimonious explanation, relative to something like "amphetamine induces a PKC signaling cascade via TAAR1 and another yet unidentified mechanism, which together account for 50% of DA efflux". If I didn't read a source that attributed a specific percentage of dopamine efflux to a protein kinase, I wouldn't have made that statement. Again, I'll look up the source for 50% of efflux via CAMKII-mediated phosphorylation if you're interested in that. In any event, I've stated above that DAT is modulated by 2 protein kinases which are activated by amphetamine via an unknown signaling cascade and which are unrelated to TAAR1.
In some ways, I don't even know how it is possible to ascribe a specific percentage to the different mechanisms involved, because many of the players have cooperative or permissive roles. I guess it could be argued that the mechanism for amphetamine effects is roughly 50% effects on vesicles and 50% effects on DAT because both of those events are required for amphetamine-induced dopamine release. But TAAR1 is not the exclusive mechanism for amphetamine effects on DAT, meaning that TAAR1 cannot be responsible for 50% of the total effect of amphetamine.
Amphetamine's effects on VMAT2 unambiguously modulate the magnitude of dopamine release through DAT. Ignoring the effects of cytosolic dopamine signaling through TAAR1, there is no known mechanism that connects a diarrhea of dopamine into the cytosol by VMAT2 to DAT phosphorylation and consequently DA efflux at the plasma membrane. When I make a statement that PKC accounts for 50% of efflux, I'm talking more about the relative amount of DA efflux for a given
preloaded concentration of DA that is dumped from VMAT2. If amphetamine had no effect on VMAT2, it would still phosphorylate DAT and produce DA efflux through DAT, but the amount of DA would be greatly reduced. All else equal, I'd suspect that PKC would still account for 50% of total efflux (the absolute amount of which would be greatly reduced) in such circumstances - the
absolute amount of effluxed DA which was mediated by PKC would change but the
relative amount (50% ) would remain fixed. If I'm still not making sense, basically what I'm saying mathematically is that if 30000 DA molecules are dumped via VMAT2 into the cytosol under normal circumstances, and amphetamine effluxes 20000 of those in total, PKC-phosphorylation of DAT is responsible for 10000 molecules being effluxed. If the effect on VMAT2 were inhibited to 1/3rd of that, the amount of efflux mediated by PKC would proportionately drop to say 3300 DA molecules.
On a completely unrelated note, I'm wondering if anyone will be eating popcorn while reading this sentence at some point.
