Ponderings and speculations about purinergic signaling, in pursuit of a unified ME/CFS theory

[frozenborderline]

This is where following random advice from the web may not be the best. I work with 2 excellent doctors who I trust and who are up on all the latest research and are in contact with other experts and who know me and prescribe treatments that are right for me.

We all need to do what is appropriate for our genes, environmental factors and health status.

And, with that said, generic studies not done on patients like us may not give accurate info in what works and what doesn't and why.

You're doing a great job of investigating various concepts ... Keep up the good work!

I guess I'm not saying your doctor is wrong, I'm just saying that IVing ATP carries more risks than supplementing intermediates or cofactors that feed the kreb's cycle. i would assume? that the kreb's cycle has an aspect of being rate limited, so over feeding it with one thing isn't necessarily like the biggest deal, but IV ATP is your bodies energy currency, and taking it directly is ballsy. I guess I'm pointing that out as much for the people here reading as anything... although it would be hard to obtain IV-able ATP anyway... But, like, I would have to trust my doctor with my life to try that, and I hope you do!

 

[frozenborderline]

This is where following random advice from the web may not be the best. I work with 2 excellent doctors who I trust and who are up on all the latest research and are in contact with other experts and who know me and prescribe treatments that are right for me.

We all need to do what is appropriate for our genes, environmental factors and health status.

And, with that said, generic studies not done on patients like us may not give accurate info in what works and what doesn't and why.

You're doing a great job of investigating various concepts ... Keep up the good work!

I guess I'm not saying your doctor is wrong, I'm just saying that IVing ATP carries more risks than supplementing intermediates or cofactors that feed the kreb's cycle. i would assume? that the kreb's cycle has an aspect of being rate limited, so over feeding it with one thing isn't necessarily like the biggest deal, but IV ATP is your bodies energy currency, and taking it directly is ballsy. I guess I'm pointing that out as much for the people here reading as anything... although it would be hard to obtain IV-able ATP anyway... But, like, I would have to trust my doctor with my life to try that, and I hope you do!

 

[Learner1]

Well, he mentioned AMP, not ATP. Its not available in the US, but if I wanted to try it, I could easily get it in Vancouver. Fortunately, we've figured out other ways of boosting my energy.

 

[frozenborderline]

oh, AMP. I saw that but thought it was a typo... I'll have to look into that.

 

[frozenborderline]

"

I think CFS is likely to involve “reductive stress,” producing lactate because pyruvate isn’t being oxidized fast enough, and in that case I think the reducing supplements, NADH and ubiquinol, could make it worse. Ubiquinone and niacinamide seem more appropriate, since they are the oxidizing forms. Drinking alcohol shifts cells to the reducing side, and a B vitamin deficiency can have similar effects. Vitamin D, thyroid, aspirin, and pregnenolone help to maintain mitochondrial oxidation.

Show original message"
--this is an email from ray peat. i've heard speculation that cfs involves oxidative stress, but ray thinks it involves "reductive stress" . this distinction is important for which supplements to get

 

[Murph]

The other day I checked Naviaux' data for ATP and adenosine. ATP was normal but adenosine was low. Today I went back to have a look at AMP, which is the intermediate state between ATP and adenosine. My thinking was that maybe looking at it could tell us a bit more about exactly where any failure to breakdown eATP is occurring.

(We assume the source of AMP and Adenosine in serum is upstream ATP. If this assumption is a simplifying assumption and it seems fair but if not please let me know.)

AMP shows an interesting difference between patients and controls with a p value of 0.001. But the relative difference between female patients and controls is actually smaller in AMP than in Adenosine.

Here's the story, starting at the beginning with ATP, same as shown earlier.

ATP levels are the same, basically. The data is only available for Women. Patients are on the left of the chart in blue ,controls in red. At the bottom of the chart I give the levels and p values (two-tailed).

AMP, however, shows real differences, (including very big differences in men driven by very high variation.) (women on left in blue, controls in red, male patients in green, male controls purple.) This is a hint that we're not breaking down ATP to AMP

Adenosine, the purine that dampens the immune response, also shows big consistent differences. ME/CFS patients are low in it. (nb. The three male controls with the highest levels of adenosine are also the three with the highest levels of AMP in the previous chart.)

I also checked on this metabolite which is a basic purine building block. nothing to report.

Why are levels of AMP so low? I wanted to see if I could use the data to check if ATP is being broken down to AMP.

I made this scatter plot of female patients showing a tantalising but weak negative correlation between them (-0.1). It *might* suggest that maybe people who build up high levels of eATP can't break them down into AMP ( it *really* looks like that if you're using your confirmation bias goggles but I remembered to take mine off just in time.)

Here's the equivalent scatter plot for the controls. As you see, not *so* different. (the slope on the line is 0.0006)

So maybe we have some very weak evidence for a failure in turning ATP into AMP. But we still need to explain the shortage of Adenosine. Let's look at the next step.

The next phase of the process is breaking down from AMP to adenosine. The relationshp between the levels looks fairly similar in patients and controls. The line of fit has roughly the same slope, suggesting maybe patients with ample AMP can make ample adenosine?

Female patients:

Female controls:

The shortage of adenosine can *probably* best be explained by a shortage of AMP, rather than a second problem in the process of breaking it down. (Although if you only look at variation of people with around 50,0000 to 60,000 level of AMP you see a big difference. Additional AMP above about 55000 seems to result in much higher levels of adenosine, suggesting a possible non-linear relationship).

Occams razor says one problem more likely than two. The big reason for the shortage of the immune-quelling particle adenosine is probably the shortage of AMP, not problems breaking AMP down.


--
To conclude. I tried to answer the question of whether ATP was higher in patients than controls. It doesn't seem to be. I tried to answer whether other (more immuno-suppressive) purines AMP and adenosine were lower in patients than controls (they seem to be).

I tried to see if we can find where the process of dephosphorylation of ATP was broken. I found a slight but curious possible negative link from ATP and AMP. And not much to report on the relatonship between AMP and Adenosine.

It all fits (albeit weakly) with a failure in cd39, and could suggest AMP supplementation as a possible route for more exploration. More data needed!

 

[Learner1]

Fascinating!

It certainly seems to help red sea bream:

https://www.ncbi.nlm.nih.gov/pubmed/27514786

 

[Murph]

Pondering also on the role of ampk: adenosine monophopshate kinase.

Most people probably figured this out already but I'm only seeing now that ampk could be the bridge between the purinergic signalling /cell danger response line of thought, and the metabolic, PDK, mTor line of thought.

AMPK is turned on when we exercise, it senses the amp to atp ratio, and it controls metabolic responses. It expects to find more amp when we exercise. If it finds less amp than expected and more atp, who knows what it might do ('buddy, you're obviously sick, stop exercising!').

(nb atp levels being discussed in this thread are outside the cell and I think ampk works inside the cell.)

If anyone more educated would like to school me on the purinergic signalling -> ampk pathway I'd sure be interested.

EDIT: One of the fun things about this disease is having the same exciting novel realisation more than once. Apparently.

 

[frozenborderline]

god, the stuff you are talking is over my head, at least on a bad day...
i'm going to try some tiny doses of naltrexone and see if i can get better enough to put more of these pieces together

 

[frozenborderline]

http://www.sciencedirect.com/science/article/pii/S2213231714000421 here's some more stuff on reductive stress

 

[anni66]

Pondering also on the role of ampk: adenosine monophopshate kinase.

Most people probably figured this out already but I'm only seeing now that ampk could be the bridge between the purinergic signalling /cell danger response line of thought, and the metabolic, PDK, mTor line of thought.

AMPK is turned on when we exercise, it senses the amp to atp ratio, and it controls metabolic responses. It expects to find more amp when we exercise. If it finds less amp than expected and more atp, who knows what it might do ('buddy, you're obviously sick, stop exercising!').

(nb atp levels being discussed in this thread are outside the cell and I think ampk works inside the cell.)

If anyone more educated would like to school me on the purinergic signalling -> ampk pathway I'd sure be interested.

EDIT: One of the fun things about this disease is having the same exciting novel realisation more than once. Apparently.

View attachment 24963

Ampk is also turned on when ATP is low

 

[pattismith]

AMP, however, shows real differences, (including very big differences in men driven by very high variation.) (women on left in blue, controls in red, male patients in green, male controls purple.) This is a hint that we're not breaking down ATP to AMP

View attachment 24953

Thank you Murph, it's amazing, the female control is so low, almost as low as ME Males!!!
Could it be an explanation about the higher prevalence of the disease in women??

 

[Murph]

Thank you Murph, it's amazing, the female control is so low, almost as low as ME Males!!!
Could it be an explanation about the higher prevalence of the disease in women??

I'm not sure what it means except that any study that doesn't analyse men and women separately risks missing something...

 

[Murph]

The purinergic signalling - AMPK link gets some good treatment in this detailed paper from 2014.

Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels – pivotal roles in hemichannel-mediated cell injury

It says that if eATP is not being broken down, AMPK is suppressed. They use suramin in a bunch of their experiments and get good results.

Extracellular ATP suppresses AMPK activation

To establish the role of the extracellular ATP, we examined the influence of ATP-degrading enzymes or exogenously added ATP on AMPK activation. As shown in Fig. 3D, the promotion of ATP degradation with Apyrase augmented AMPK activation, whereas supplementing with ATP counteracted the effect of hemichannel blockers on AMPK activation (Fig. 3E,F). These results indicate that hemichannel-derived ATP suppresses AMPK activation.

Most of the cellular actions of extracellular ATP are mediated through its binding to P2 purinoceptors (Baroja-Mazo et al., 2013; Wang et al., 2013). At present, two classes of purinoceptors have been identified (P2X and P2Y) and both are expressed in renal tubular cells (Praetorius and Leipziger, 2010). We therefore examined the possible involvement of purinergic signaling in the suppression of AMPK activation. Suramin and PPADS, two effective antagonists of P2 purinoceptors (Charlton et al., 1996), markedly potentiated AMPK activation, indicating an involvement of purinergic signaling in suppression of AMPK activation (Fig. 3G,H).

They also talk a lot about how hemichannels depend on calcium concentrations, which made me think of the work from Griffith university.

Download the full paper here: https://sci-hub.io/10.1242/jcs.139089

 

[Murph]

@necessary8

Janet confirmed to me on twitter that the ATP that Ron used to return the assay to normal was extracellular.

https://twitter.com/i/web/status/927710816731742208

she emphasised that this was a preliminary experiment that had been done only once.

It's a mystery precisely how this fits with purinergic signalling.

 

[nandixon]

@necessary8

Janet confirmed to me on twitter that the ATP that Ron used to return the assay to normal was extracellular.

Thanks for confirming this. I had mistakenly been thinking the ATP was added intracellularly. This raises some very interesting questions and possibilities.

First, when exogenous ATP is added extracellularly many things happen, but one of the most fundamental is that it causes an influx of calcium ions (Ca2+) into the cell. This happens via ATP’s effect on the purinergic 2X receptors, e.g., P2X7:

Activation of the P2X7 receptor initiates a series of cellular responses that include depolarization, activation of phospholipase C, and a rise in intracellular Ca2+ concentrations, which stimulates caspase-1 activity, cytokine release, and activation of p38 mitogen-activated protein kinase (MAPK) (Armstrong et al. 2002; Ferrari et al. 2006; Donnelly-Roberts et al. 2009). Furthermore, activation of P2X7 leads to recruitment, binding and activation of additional channel-forming proteins, namely, the glycoprotein pannexin 1(Locovei et al. 2007). Such events, in turn, have a plethora of effects. For example, cytokine signaling provokes an inflammatory stimulus in cells of the immune system (Volonte et al. 2012)...[Ref 2015]

Likewise for the activation of T cells:

The binding of extracellular ATP to P2X receptors, which are ATP-gated ion channels, induces the influx of extracellular Ca2+ (9, 10). Among the seven known P2X receptor subtypes (P2X1–7), P2X7 receptors are particularly highly expressed in immune tissues (9,10,11,12,13). In T cells, P2X7 receptors initiate Ca2+-dependent downstream signals that lead to T-cell activation and proliferation (4, 14). Activation of P2X7 receptors has also been implicated in the production of proinflammatory cytokines (e.g., IL-2, IL-1β, and IL-18) and in the regulation of cell proliferation and cell death (14,15,16,17,18). In human peripheral blood mononuclear cells (PBMCs) and T cells, P2X7 receptor stimulation induces proliferation and IL-2 synthesis (4, 14, 19), while pharmacological blockade of P2X7 receptors inhibits TCR-stimulated T-cell activation (18). [Ref 2009]

As @Murph suggested, it seems inexplicable that added extracellular ATP or suramin can both appear to normalize the ME/CFS cellular reading using Dr Ron Davis’ nano-needle chip device, because they are seemingly doing the opposite of the other:

To evaluate the role of P2X receptors in the influx of Ca2+ and activation of IL-2 expression, we used pharmacological inhibitors to block P2X receptors. The P2X receptor antagonists suramin and NF023 block a number of P2X receptor subtypes, while o-ATP [oxidized ATP] is a more selective inhibitor of P2X7 receptors (11, 36, 37). We found that suramin (100 μM), NF023 (10 μM), and o-ATP (10 μM) inhibited Ca2+ mobilization of Jurkat cells stimulated with anti-CD3 antibodies (1 μg/ml) or exogenous ATP (2 mM; Fig. 4A, B). Treatment of human PBMCs with suramin (100 and 500 μM) or o-ATP (10 and 100 μM) inhibited IL-2 production (Fig. 4C). [Same ref as preceding]

There are probably a number of ways to potentially reconcile the nano-needle results, but one simple way would be that it may be that for a given concentration of ATP or suramin that the different P2 receptors may be differentially activated or inhibited. I think that suramin, for example, may have a weaker antagonistic effect at P2X7 than at some of the other P2 receptors.

Anyway, I mostly mentioned all of the above just for some background, because it seems that it might be possible to emulate, in vivo, the effect that is seen when extra ATP is added extracellularly in vitro by using an older over-the-counter antihistamine called clemastine based on the following 2011 study:

Clemastine Potentiates the Human P2X7 Receptor by Sensitizing It to Lower ATP Concentrations

To assess the impact of pharmaceuticals on P2X7, we screened a compound library comprising approved or clinically tested drugs and identified several compounds that augment the ATP-triggered P2X7 activity in a stably transfected HEK293 cell line. Of these, clemastine markedly sensitized Ca2+ entry through P2X7 to lower ATP concentrations. Extracellularly but not intracellularly applied clemastine rapidly and reversibly augmented P2X7-mediated whole-cell currents evoked by non-saturating ATP concentrations. Clemastine also accelerated the ATP-induced pore formation and Yo-Pro-1 uptake, increased the fractional NMDG+ permeability, and stabilized the open channel conformation of P2X7. Thus, clemastine is an extracellularly binding allosteric modulator of P2X7 that sensitizes P2X7 to lower ATP concentrations and facilitates its pore dilation. The activity of clemastine on native P2X7 receptors, Ca2+ entry, and whole-cell currents was confirmed in human monocyte-derived macrophages.

Clemastine is often a sedating type of antihistamine so might have to be taken at night. There are only a handful of posts about it on the Phoenix Rising forum, including this recent one a couple weeks ago:

Allergy Drug Improves Function in Patients with Chronic Injury from Multiple Sclerosis

Seems pretty unlikely that any OTC drug that's been on the market for a number of years wouldn't have already been discovered as a treatment by ME/CFS patients but… I'm off to the store to buy some right now.

 

[frozenborderline]

https://www.ncbi.nlm.nih.gov/pubmed/17391109
has anyone here heard of ethyl pyruvate?

 

[frozenborderline]

Thanks for confirming this. I had mistakenly been thinking the ATP was added intracellularly. This raises some very interesting questions and possibilities.

First, when exogenous ATP is added extracellularly many things happen, but one of the most fundamental is that it causes an influx of calcium ions (Ca2+) into the cell. This happens via ATP’s effect on the purinergic 2X receptors, e.g., P2X7:




Likewise for the activation of T cells:




As @Murph suggested, it seems inexplicable that added extracellular ATP or suramin can both appear to normalize the ME/CFS cellular reading using Dr Ron Davis’ nano-needle chip device, because they are seemingly doing the opposite of the other:




There are probably a number of ways to potentially reconcile the nano-needle results, but one simple way would be that it may be that for a given concentration of ATP or suramin that the different P2 receptors may be differentially activated or inhibited. I think that suramin, for example, may have a weaker antagonistic effect at P2X7 than at some of the other P2 receptors.

Anyway, I mostly mentioned all of the above just for some background, because it seems that it might be possible to emulate, in vivo, the effect that is seen when extra ATP is added extracellularly in vitro by using an older over-the-counter antihistamine called clemastine based on the following 2011 study:

Clemastine Potentiates the Human P2X7 Receptor by Sensitizing It to Lower ATP Concentrations




Clemastine is often a sedating type of antihistamine so might have to be taken at night. There are only a handful of posts about it on the Phoenix Rising forum, including this recent one a couple weeks ago:

Allergy Drug Improves Function in Patients with Chronic Injury from Multiple Sclerosis

Seems pretty unlikely that any OTC drug that's been on the market for a number of years wouldn't have already been discovered as a treatment by ME/CFS patients but… I'm off to the store to buy some right now.

let us know how your bioassay goes!

 

[Murph]

Clemastine is often a sedating type of antihistamine so might have to be taken at night.… I'm off to the store to buy some right now.

Sweet dreams @nandixon! report back please.

Although if the positive effect happens while you're knocked out, how will we know!?

 

[dreampop]

Thanks for confirming this. I had mistakenly been thinking the ATP was added intracellularly. This raises some very interesting questions and possibilities.

@necessary8 gave an explanation on the first page in his replies.