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

[frozenborderline]

@Murph, I keep coming back to phosphorylation and low adenosine - so I'm back on the coffee I've been reading the old original potato apyrase experiments which talk about ATP and the way it competitively interacts with ADP, AMP and adenosine. I wondered if this is the metabolic trap Phair was talking about - ATP getting in the way of other metabolic processes which require phosphorylation (such as ubiquinone), particularly at the PANX 1 channel. So as a little experiment, I started using glycyrrhetinic acid (licorice extract) as a PANX1 inhibitor - to slow down ATP into the extracellular space - and hence the p2x7 responsiveness. It's been a week (and I've started slow) I have less lactic acid and much more energy. I had a flare of what I think of as p2x3 responses - wide spread pain, trigeminal nerve issues, gut distention issues ect ect. I'm taking ALA for that but it takes awhile to take effect. All over, I'm feeling much better.

I forget the stuff I was looking at but it's my understanding that glycyrrhetinic acid does something with cortisol and aldosterone that often makes blood pressure rise dramatically, and that this can be long lasting. Very curious about your experience on it though.

 

[pattismith]

This adenosine is certainly an important key.

Adenosine is metabolized into Inosine by deaminase enzyme, then catabolized into uric acid inside the cells.

Uric acid has neurologic anti oxidant properties, and was found to be inversely correlated with outcome in MS and ALS, so I would be interested if we can find the same in CFS/ME!

Inosine can actively binds to adenosine receptors A1 and A2A , so I guess the fact CFS/ME doctors gives inosine to their patients may be to compensate the adenosine deficiency.


I opened a thread about it here
https://forums.phoenixrising.me/ind...-anti-neurogenic-detrusor-overactivity.62212/

I didn't investigate the purinergic signaling /low adenosine link, but I found this study about Duchenne muscular dystrophy (below).

This study is very interesting because Allopurinol is blocking the Xanthine Oxidase, which is supposed to stop the Inosine catabolism into Uric acid (Allopurinol is a drug used to lower blood uric acid in gout patients).


Purine and carnitine metabolism in muscle of patients with Duchenne muscular dystrophy


Abstract
We determined levels of purines, purine metabolites, related enzymes and carnitine in muscle of 8 untreated Duchenne muscular dystrophy (DMD) patients, 12 allopurinol-treated DMD patients and 12 age-matched controls. Muscle of DMD patients was found to be deficient in ATP, ADP, adenylsuccinate, hypoxanthine, guanine and adenylsuccinate synthetase.

In allopurinol-treated DMD patients, mean total adenylate level was only three times less than in controls (versus 14 times less in untreated DMD patients).

Mean inosine monophosphate (IMP), adenine, adenosine, inosine, xanthine, guanine, guanosine and uric acid levels were higher in allopurinol-treated patients than in controls, while mean adenylsuccinate levels were higher than in untreated patients.

Allopurinol also restored acylcarnitine levels to normal and significantly increased free carnitine levels.
These findings strongly support the hypothesis that Duchenne muscular dystrophy involves alterations leading to blockage of the IMP → purine pathway and that allopurinol treatment favours restoration of purine levels by this route.

Furthermore, our results suggest that the observed deficiencies in cell components unrelated to purine metabolism are long-term secondary effects.

However, another purine was one of the clearest and most consistent results: adenosine.

View attachment 24895


As you can see here adenosine levels were within a more narrow range and quite different on average between patients and controls. I haven't done p values on this but I guess I should.

View attachment 24897

Adenosine certainly has a range of interesting roles.

View attachment 24896

I'm personally always interested in vasodilation/vasocontraction problems (because of POTS, widespread problems with alcohol, the Fluge Mella NO patent, etc.). I think endothelial cells, being bloodflow facing immune signalling cells, are a perfect suspect that might decide to turn a local problem (lack of appropriate vascular tone) into a global one: an acute systemic immune response. Which could be a great explanation for PEM.

Adenosine is also tied up in metabolism, of course.

View attachment 24898

I'm certainly interested to hear if anyone can find any strong links from Adenosine to the puringeric signalling theory!

EDIT: this paper may be interesting:
Extracellular Adenosine-Mediated Modulation of Regulatory T Cells

 

[frozenborderline]

https://www.ncbi.nlm.nih.gov/pubmed/25695577/

Lidocaine is a p2x7 antagonist, which is very interesting. The controversial cfs doc jay Goldstein used intravenous lidocaine in ME/CFS patients as one of his most used treatments.

 

[frozenborderline]

“The transmitter ATP, usually coreleased with NE, is extremely nocicep- tive and has been implicated in the pathophysiology of reflex sympathetic dystrophy, also known as complex regional pain syndrome. It is released from microvascular endothelial cells during migraine headache and angina. ATP release is associated with distension of the tubular viscera (Burnstock G, 2001). There are two classes of purine receptors, P1 (adenosine) and P2X (mostly for ATP). The P2X2 and P2X3 receptors can be coexpressed to pro- duce a channel with a slowly desensitizing response. P2X3 receptors are not affected by pH, but recombinant P2X2 receptors are strongly pH sensitive. The combination of the two is still pH sensitive but to a lesser extent (Stoop R et al., 1997). Acid pH augments the excitatory actions of ATP on dissociated mammalian sensory neurons. Because prescribing suramin, a general an- tagonist of all PX receptors, is not feasible, and receptor-specific antago- nists are not yet available, consideration should be given to mild alkalini- zation of patients with painful neurosomatic disorders. The response is prompt, and the alkalinizing agent may be titrated by tolerance and effec- tiveness.” From Goldstein’s “tuning the brain”. So he is suggesting either other hypothetical purinergic antagonists which could be safer than suramin or altering ph to affect purinergic receptors

 

[pattismith]

....
P2X7 Antagonists:
....

• Colchicine inhibits P2X7 receptor-associated pore opening at EC50 = 290 to 540 μM. Ref: 1 Prof Jose Montoya uses colchicine as an ME/CFS treatment.

....

Colchicine is probably our best chance to improve (cheap and old drug).
Not by P2X7 inhibition though, but by Caspase-1 inhibition which is the key to reduce neuro-inflammation.
Side effects must be monitored for long lasting treatment.

Fig. 1: Virus-intrinsic and host-intrinsic mechanisms of inflammasome activation. | Nature Reviews Immunology

 

[Marylib]

Hopefully. I know an ampligen responder (no longer receiving it) who tried colchicine when she was flattened and developed new neurological symptoms after one dose of the pfizer vaccine. So maybe that additional inflammatory response wiped out what gains she might have had on colchicine? Or her body accepted that S1 protein as a SARS Co V2 infection?

 

[Learner1]

Colchicine is probably our best chance to improve (cheap and old drug).
Not by P2X7 inhibition though, but by Caspase-1 inhibition which is the key to reduce neuro-inflammation.

LDN can reduce neuroinflammation in microglial cells, which are the immune system in the brain.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962576/

 

[pattismith]

LDN can reduce neuroinflammation in microglial cells, which are the immune system in the brain.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962576/

If Naltrexone (TLR2 and TLR4 inhibitor) doesn't work, colchicine (caspase 1 inhibitor) is another option.
In auto-inflammatory diseases where Colchicine is indicated, it's recognised that even a small dose of colchicine can have beneficial effect.
Even a pulsed low dosage may be beneficial for those who can't tolerate colchicine.

Toll-like receptors, the NLRP3 inflammasome, and interleukin-1β in the development and progression of type 1 diabetes | Pediatric Research (nature.com)

 

[Violeta]

I'm working my way through the 10 pages of this thread, but may I ask if anyone has found a low purine diet helpful?

 

[Learner1]

I'm working my way through the 10 pages of this thread, but may I ask if anyone has found a low purine diet helpful?

A low purine diet would be devastating for me, loaded with gluten, dairy, eggs and oxalates.

 

[Hip]

Two studies which find high P2X7 receptor expression and activation in long COVID:

https://www.nature.com/articles/s41380-020-00965-3
https://www.biorxiv.org/content/10.1101/2022.03.04.483019v1

The first paper says:

P2X7 receptor antagonism could be a promising strategy to prevent or treat neurological complications in COVID-19 patients

 

[Murph]

THis thread's old and dead but here's a weird little study that provides a tiny shred of evidence that low cd73 activity might not be a problem anyway.

Deletion of CD73 increases exercise power in mice​

Aderbal S Aguiar Jr 1 2 , Ana Elisa Speck 3 4 , Paula M Canas 3 , Rodrigo A Cunha 3 5

DOI: 10.1007/s11302-021-09797-4

Abstract​

Ecto-5'-nucleotidase or CD73 is the main source of extracellular adenosine involved in the activation of adenosine A2A receptors, responsible for the ergogenic effects of caffeine. We now investigated the role of CD73 in exercise by comparing female wild-type (WT) and CD73 knockout (KO) mice in a treadmill-graded test to evaluate running power, oxygen uptake (V̇O2), and respiratory exchange ratio (RER) - the gold standards characterizing physical performance. Spontaneous locomotion in the open field and submaximal running power and V̇O2 in the treadmill were similar between CD73-KO and WT mice; V̇O2max also demonstrated equivalent aerobic power, but CD73-KO mice displayed a 43.7 ± 4.2% larger critical power (large effect size, P < 0.05) and 3.8 ± 0.4% increase of maximum RER (small effect size, P < 0.05). Thus, KO of CD73 was ergogenic; i.e., it increased physical performance.

 

[Murph]

A new paper from the OMF outpost here in Melbourne Australia investigates purinergic sgnalling.

Frequencies and expression of purinergic pathway markers on B cells from HC and ME/CFS patients​

A series of ectoenzymes regulate the extracellular purinergic signaling pathways through their ability to modulate the levels of ATP and adenosine. Degradation to adenosine is associated with the activation of anti-inflammatory pathways. We therefore followed the frequencies and MFI (expression) of the ectonucleotidases CD39 and CD73 and of the NAD-degrading enzyme cyclic ADP ribose hydrolase (CD38) on live B cells cultured from HC and ME/CFS patients (Figure 2).

The results from each stimulus were combined due to low cell numbers. At baseline, these three surface markers were found in similar frequencies in both HC and ME/CFS patients with no differences in expression. During culture, however, the frequencies (%) of both CD38+ and CD73+ B cells were significantly higher in ME/CFS compared with HC cultures (Figures 2A, C).

The mean expression (MFI) of CD38, but not CD73, was found to be significantly increased in cultures from ME/CFS patients compared with HC at days 1 and 3, which corresponds with the period of most proliferation (Figures 2D, F). The expression (MFI) of CD39 was not different between patients and controls (Figure 2E).

https://www.frontiersin.org/article...=EMLX&utm_campaign=PRD_FEOPS_20170000_ARTICLE

Purinergic signalling is not really the main part of the paper though. It's principally about b-cell metabolism, showing they use different substrates to make ATP. They foiund that if they left b-cells alone in a petri dish they ate all the essential amino acids up and the amount left in the dish was small. This all feeds into the Melbourne model of mecfs which recommends amino acid supplementation (which I'm on and experience benefit from. They could even be my b-cells floating round in there so it's possible my affirmation is not an independent support of the findings!)

 

[SlamDancin]

@Murph i think they finally published the final version of the abstract, which also includes CD24

Results: Proliferating B cells from patients with ME/CFS showed a lower mitochondrial mass and a significantly increased usage of essential amino acids compared with those from HC, with a significantly delayed loss of CD24 and an increased expression of CD38 following stimulation.

Discussion: The immunophenotype results suggested the triggering of a stress response in ME/CFS B cells associated with the increased usage of additional substrates to maintain necessary ATP levels. Disturbances in energy metabolism in ME/CFS B cells were thus confirmed in a dynamic in vitro model, providing the basis for further mechanistic investigations.

Am I correct in understanding that CD24 is more or less a marker of B cell activation?

Also which AA’s do you take? Thanks 🙏

A question for everyone, would Niacinamide supplementation help provide NAD?

 

[lyran]

A question for everyone, would Niacinamide supplementation help provide NAD?

Yes if the pathway is working correctly.

 

[datadragon]

ZINC...IS THE LINK! Zinc deficiency causes delayed ATP clearance and adenosine generation https://forums.phoenixrising.me/threads/role-of-dysfunctional-glycolysis.91264/post-2449814

Extracellular ATP can cause P2X receptors to activate the NOD-like receptor 3 (NLRP3) inflammasome and cause IL-1β and IL-18 maturation and release https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052171/
https://pubmed.ncbi.nlm.nih.gov/23434541/ This causes lowered zinc availability and uptake as one downstream effect. NLRP3 inflammasome activation leads to endoplasmic reticulum stress which causes high WASF3 levels and disrupts Mitochondrial function, while blocking ER stress lowered WASF3 levels and restored mitochondrial function.

Zinc deficiency evokes the endoplasmic reticulum (ER)-stress response https://pubmed.ncbi.nlm.nih.gov/23748779/ https://forums.phoenixrising.me/thr...possible-treatment-for-cfs.37244/post-2451850 Butyrate inhibits ER stress as mentioned but also zinc https://pubmed.ncbi.nlm.nih.gov/32549180

P2X7 receptor (P2X7R) is required for secretion of IL-1, and can be blocked by divalent cations such as magnesium (Mg). We demonstrated that Magnesium sulfate is efficacious in blocking IL-1-mediated-inflammation in HUVECs via downregulation of P2X7Rs on HUVECs https://pubmed.ncbi.nlm.nih.gov/31493768/

The marginal vitamin B6 deficiency appears to relate to an increased risk of inflammation-related diseases, Recent studies have revealed that vitamin B6 treatment increases cardiac levels of imidazole dipeptides (eg, carnosine, anserine, and homocarnosine), histamine, and -aminobutyric acid (GABA) and suppresses P2X7 receptor-mediated NLRP3 inflammasome. These modulations may implicate possible cardioprotective mechanisms of vitamin B6. These modulations may also be involved in the underlying mechanisms through which vitamin B6 suppresses oxidative stress and inflammation. (the active pyridoxal 5 phosphate (P5P) NOT pyridoxine which lowers active b6) https://link.springer.com/article/10.1007/s00394-021-02665-2

The active form of Vitamin B6 (P5p) prevents IL-1β production by inhibiting NLRP3 inflammasome activation and suggest its potential for preventing inflammatory diseases driven by the NLRP3 inflammasome. https://pubmed.ncbi.nlm.nih.gov/27733681/

Note that vitamin B6 requires zinc in both its metabolism to the active form and entry to the cell https://forums.phoenixrising.me/thr...6-nk-cells-histamine-oh-my.90803/post-2443820 Again I see a see saw in all the areas we have been looking at revolving around zinc status.

Most of the main problems being discussed happen together at the same time due to the zinc changes that happen from the presence of pro inflammatory cytokines under inflammation/infection, and are not all separate things to deal with individually. That does not mean there is no possibility of other deficiencies beyond that or problems on top of that, just that this is the core issue that can cause problems across the board. In other words to simplify, inflammation/infection is lowering zinc availability and uptake, and the zinc unavailability happens to have huge numbers of downstream effects.

 

[Murph]

@Murph

Also which AA’s do you take? Thanks 🙏

A question for everyone, would Niacinamide supplementation help provide NAD?

I take unflavoured hydrolysed whey protein isolate.
this is an example: https://www.amazon.com.au/gp/product/B09LSWFL5Q/ref=ewc_pr_img_1?smid=A2GUZBG4F53PKQ&psc=1 but I have tried many brands e.g https://www.bulknutrients.com.au/products/whey-protein-isolate.

I'm told hydroysed whey protein isolate is absorbed the quickest so I buy that even though whey protein concentrate is cheaper.

It's possible my body needs only one or two of the aminos in there but I don't know which! So i just take them all.

I've been taking very large amounts for years whenever I do exercise. it is immediately noticeable in terms of its effect in dodging fatigue during exercise, and it also correlates with less PEM later. But we're not talking teaspoonfuls. I might have 100g with exercise.

I do worry i might have harmed my kidneys by eating so much protein. I expect it has kept my weight higher than it would otherwise be too.
-
As for NAD, I recently read a book by a longevity guru called David SInclair. He's a harvard professor but also seems a bit starry-eyed and I certainly don't believe everything he says. Nevertheless he recommends a NAD precursor called NMN (Nicotinamide mononucleotide).

Some research is starting out on that and while it's early days, the evidence is certainly not against it. it has made a lot of mice feel better and the early studies on people show some promise:
https://pubmed.ncbi.nlm.nih.gov/36482258/

however some people think nicotinamide ribose might be a better choice than NMN: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10721522/

 

[SlamDancin]

Thank you @Murph I was just about to buy a Whey isolate so that’s great to hear. Whey is healthy and probably healthier than the aminos separately.

I’ve seen many studies on NMN that *seem* to suggest that it can legitimately raise NAD intercellularly and intracellularly, although it’s dose dependent which gets expensive. Niacinamide may be more cost effective.

 

[datadragon]

Vitamin B6 deficiency also lowers NAD since conversion is impaired.

About 95% of TRP is metabolized through the kynurenine )KYN) pathway to produce NAD.
TRP- or indoleamine-2,3-dioxygenases (TDO or IDO) convert TRP to KYN and the activity of these enzymes is a rate-limiting step, increased by stress hormones or inflammatory factors (e.g., IFNG and LPS). KYN is then converted in 3-hydroxykynurenine (3-HKYN), through the action of KYN-monooxygenase (KMO). KYN and
3-HKYN can be converted, respectively, in kynurenic acid (KYNA) and xanthurenic acid (XA), through the activity of the aminotransferases (KAT), which is a PLP-dependent enzyme. The conversion of 3-HKYN into the 3-hydroxyanthranilic acid (3-HAA) is performed by kynureninase (KYNU), which also depends on PLP for its activity. As KYNU is more sensitive to deficiency of PLP, with respect to KAT, PLP deficiency diverts 3-HKYN metabolism from the formation of 3-HAA, to accumulation of KYNA and XA (and away from NAD). https://www.researchgate.net/public...iabetes_Relationship_and_Molecular_Mechanisms

 

[Hip]

In other words to simplify, inflammation/infection is lowering zinc availability and uptake, and the zinc unavailability happens to have huge numbers of downstream effects.

Your whole post sounds like pseudoscience, attempting to explain the ill effects of infection and inflammation in the body in terms of zinc deficiency.

The body is not that simple; the adverse effects experienced in various illnesses do not revolve around nutrient deficiencies, as you seem to be suggesting.

Many diseases involve chronic inflammation, yet zinc supplementation does not do much for them.