http://www.blackwell-synergy.com/doi/abs/10.1111/j.1471-4159.1993.tb03497.x

Increased Acetylcholine Content Induced by Adenosine in a Sympathetic Ganglion and Its Subsequent Mobilization by Electrical Stimulation
A. Tandon11Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, CanadaB. Collier11Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada1Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
Address correspondence and reprint requests to A. Tandon at Department of Pharmacology and Therapeutics, McGill University, 3665 Drummond St., Montreal, Quebec, Canada, H3G 1Y6.
ACh, acetylcholine; Appcp, βγ-methylene-adenosine 5'-triphosphate; ChAT, choline acetyltransferase; HC-3, hemicholinium-3; meclonazepam, (S)-5-(2-chlorophenyl)-1,3-dihydro-3-methyl-7-nitro-2H-1,4-benzodiazepin-2-one; NBTI, nitrobenzylthioinosine; PCh, phosphorylcholine; Ro 11-3624, (R)-5-(2-chlorophenyl)-1,3-dihydro-3-methyl-7-nitro-2H-1,4-benzodiazepin-2-one; TCA, trichloroacetic acid; TPB, tetraphenylboron. vesamicol, 2-(4-phenylpiperidino)cyclohexanol..

Abstract
Abstract: The present study was initiated to examine the effects of ATP [adenosine triphosphate] on acetylcholine (ACh) synthesis. The exposure of superior cervical ganglia to ATP increased ACh stores by 25%, but this effect was also evident with ADP, AMP, and adenosine, but not with βγ-methylene ATP, a nonhydrolyzable analogue of ATP, or with inosine, the deaminated product of adenosine. Thus, we attribute the enhanced ACh content caused by ATP to the presence of adenosine derived from its hydrolysis by 5'-nucleotidase. The adenosine-induced increase of tissue ACh was not the consequence of an adenosine-induced decrease of ACh release. The extra ACh remained in the tissue for more than 15 min after the removal of adenosine, but it was not apparent when ganglia were exposed to adenosine in a Ca2+-free medium. Incorporation of radiolabelled choline into [3H]ACh was also enhanced in the presence of adenosine, suggesting an extracellular source of precursor. Moreover, the synthesis of radiolabelled forms of phosphorylcholine and phospholipid was not reduced in adenosine's presence, suggesting that the extra ACh was not likely derived from choline destined for phospholipid synthesis. Aminophylline did not prevent the adenosine effect to increase ACh content; this effect was blocked by dipyridamole, but not by nitrobenzylthioinosine (NBTI). In addition, two benzodiazepine stereoisomers known to inhibit stereoselectively the NBTI-resistant nucleoside transporter displayed a similar stereoselective ability to block the effect of adenosine. Together, these results argue that adenosine is transported through an NBTI-resistant nucleoside transporter to exert an effect on ACh synthesis. The extra ACh accumulated as a result of adenosine's action was releasable during subsequent preganglionic nerve stimulation, but not in the presence of vesamicol, a vesicular ACh transporter inhibitor. We conclude that the mobilization of ACh is enhanced as a result of adenosine pretreatment.

http://grande.nal.usda.gov/ibids/index.php?mode2=detail...rences&therow=592490

Selective depletion of the acetylcholine and vasoactive intestinal polypeptide of the guinea-pig myenteric plexus by differential mobilization of distinct transmitter pools.


The effect of electrical field stimulation on the release of acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) from superfused strips of myenteric plexus-longitudinal muscle (MPLM) of guinea-pig ileum and on the transmitter content of the tissue was investigated at different frequencies and in the presence and absence of choline hemicholinium-3 and colchicine. Low frequency electrical field stimulation released ACh by more than 4 times the basal release; the simultaneously detected VIP secretion was increased only slightly above the resting level. During high frequency stimulation (50 Hz) the release of VIP was greatly increased (to 5 times the resting release) whereas the release of ACh increased to only 150% of the basal output. When choline was present, the ACh content of the tissue itself was not altered by electrical stimulation indicating a rate of synthesis sufficient to maintain release. It [acetylcholine content] was reduced in a frequency-dependent manner in the absence of exogenous choline or in the presence of 10 microM hemicholinium-3 (an inhibitor of choline uptake) by up to 54% of the original content. A similar but even larger reduction took place in the amount of ACh released. Neither the secretion of VIP nor the tissue VIP content was altered by these treatments. Long-lasting (greater than 60 min) high-frequency (50 Hz) stimulation resulted in the depletion of the VIP pool (by 25%) while the ACh content remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

http://en.wikipedia.org/wiki/Asymmetric_dimethylarginine

Asymmetric dimethylarginine (ADMA) is a naturally occurring chemical found in blood plasma. It is a metabolic by-product of continual protein modification processes in the cytoplasm of all human cells. It is closely related to L-arginine, a conditionally-essential amino acid. ADMA interferes with L-arginine in the production of nitric oxide, a key chemical to endothelial and hence cardiovascular health.

Discovery
Patrick Vallance and his London co-workers first noted the interference role for asymmetric dimethylarginine.[1] Today biochemical and clinical research continues into the role of ADMA in cardiovascular disease, diabetes mellitus, erectile dysfunction and certain forms of kidney disease.

Asymmetric dimethylarginine is created in protein methylation, a common mechanism of post-translational protein modification. This reaction is catalyzed by an enzyme set called S-adenosylmethionine protein N-methyltransferases (protein methylases I and II).[2] The methyl groups transferred to create ADMA are derived from the methyl group donor S-adenosylmethionine, an intermediate in the metabolism of homocysteine. (Homocysteine is an important blood chemical, because it is also a marker of cardiovascular disease). After synthesis, ADMA migrates into the extracellular space and thence into blood plasma. Asymmetric dimethylarginine is measured using high performance liquid chromatography.

ADMA concentrations are substantially elevated by native or oxidized LDL cholesterol.[3] Thus a spiralling effect occurs with high endothelial LDL levels causing greater ADMA values, which in turn inhibit NO production needed to promote vasodilation. The elimination of ADMA occurs through urine excretion and metabolism by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The role of homocysteine as a risk factor for cardiovascular disease is suggested to be mediated by homocysteine down-regulating production of DDAH in the body. Polyphenol antioxidants also play a role in down-regulating homocysteine.

ADMA and suggested lines of therapeutic research

With raised levels of ADMA seemingly to be associated with adverse human health consequences for cardiovascular disease, metabolic diseases and also a wide range of diseases of the elderly, the possible lowering of ADMA levels may have important therapeutic effects. However it has yet to be established whether ADMA levels can be manipulated and, more importantly, if this results in useful clinical benefits.

The association of ADMA with abnormalities of lipid regulation suggested that supplements of free fatty acids might manipulate ADMA levels. However research has failed to show that these have an effect.[4][5]

ADMA role has been linked with elevated levels of homocysteine.[6][7][8] Whilst approaches at modifying the later with oral supplements of folic acid were strongly suggested, studies have shown this fails to give any clinical benefit and suggested that B vitamins might instead increase some cardiovascular risks.[9][10][11]

Direct alteration of ADMA levels with supplements of L-arginine have been suggested.[12][13] The hope is that such intervention might not only improve endothelial function but also reduce clinical symptoms of overt cardiovascular disease.[14][15] However studies show inconsistency in results in a clinical context,[16] and the recent results with manipulating homocysteine levels warrant extreme care with what clinical outcomes might arise from this approach.

Statins, as well as affecting circulating cholesterol levels, also increase nitric oxide levels and so have a direct effect on blood supply to the heart. Elevated levels of ADMA seems to modify this effect and so may have consequences for patients' responsiveness to taking statins.[17]

This message has been edited. Last edited by: nitekitty,

http://en.wikipedia.org/wiki/Dimethylarginine_dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme found in all mammalian cells. Two isoforms exist, DDAH I and DDAH II, with some differences in tissue distribution of the two isoforms[1]). The enzyme degrades methylarginines, specifically asymmetric dimethylarginine (ADMA) and NG-monomethyl-L-arginine (MMA). The methylarginines ADMA and MMA inhibit the production of nitric oxide synthase.[2] Accordingly, DDAH is important in removing methylarginines, generated by protein degradation, from accumulating and inhibiting the generation of nitric oxide.

Inhibition of DDAH activity causes methylarginines to accumulate, blocking nitric oxide(NO) synthesis and causing vasoconstriction.[3] An impairment of DDAH activity appears to be involved in the elevation of plasma ADMA, and impairment of vascular relaxation observed in humans with cardiovascular disease or risk factors (such as hypercholesterolemia, diabetes mellitus, and insulin resistance). The activity of DDAH is impaired by oxidative stress, permitting ADMA to accumulate. A wide range of pathologic stimuli induce endothelial oxidative stress such as oxidized LDL-cholesterol, inflammatory cytokines, hyperhomocysteinemia, hyperglycemia and infectious agents. Each of these insults attenuates DDAH activity in vitro and in vivo.[4][5][6][7] The attenuation of DDAH allows ADMA to accumulate, and to block NO synthesis. The adverse effect of these stimuli can be reversed in vitro by antioxidants, which preserve the activity of DDAH.

The sensitivity of DDAH to oxidative stress is conferred by a critical sulfhydryl in the active site of the enzyme that is required for the metabolism of ADMA. This sulfhydryl can also be reversibly inhibited by NO in an elegant form of negative feedback.[8] Homocysteine (a putative cardiovascular risk factor) mounts an oxidative attack on DDAH to form a mixed disulfide, inactivating the enzyme.[5] By oxidizing a sulfhydryl moiety critical for DDAH activity, homocysteine and other risk factors cause ADMA to accumulate and to suppress nitric oxide synthase (NOS) activity.

The critical role of DDAH activity in regulating NO synthesis in vivo was demonstrated using a transgenic DDAH mouse.[9] In this animal, the activity of DDAH is increased, and plasma ADMA levels are reduced by 50%. The reduction in plasma ADMA is associated with a significant increase in NOS activity, as plasma and urinary nitrate levels are doubled. The increase in NOS activity translates into a 15mmHg reduction in systolic blood pressure in the transgenic mouse. This study provides evidence for the importance of DDAH activity and plasma ADMA levels in the regulation of NO synthesis. Subsequent studies have shown that DDAH transgenic animals also manifest improvements in endothelial regeneration and angiogenesis, and reduced vascular obstructive disease, in association with the reduced plasma levels of ADMA.[10][11] These findings are consistent with evidence from a number of groups that nitric oxide plays a critical role in vascular regeneration. By contrast, elevations in ADMA impair angiogenesis. These insights into the role of DDAH in degrading endogenous inhibitors of NOS, and thereby maintaining vascular NO production, may have important implications in vascular health and therapy for cardiovascular disease.

http://cardiovascres.oxfordjournals.org/cgi/content/full/40/1/45
Vagal nerve stimulation releases vasoactive intestinal peptide which significantly increases coronary artery blood flow

Objective: To determine the effects of vasoactive intestinal peptide (VIP), released endogenously from cardiac vagal nerves, on coronary artery blood flow (CBF). Methods: We determined the effects of vagal nerve stimulation (VNS) at frequencies of 10, 15, 20, and 30 Hz on left circumflex coronary artery (LCx) blood flow. The increases in CBF during VNS were compared with the increases in CBF produced by exogenous VIP and also nitroglycerin (NTG). In 18 anesthetized open chest mongrel dogs, we blocked the muscarinic and β-adrenergic receptors with atropine and propranolol. We controlled heart rate and aortic pressure by right atrial pacing and an arterial reservoir. CBF was measured in the LCx with a Doppler flow probe. A 25 gauge catheter was placed in the proximal LCx to inject the VIP receptor antagonist [4Cl-D-Phe6Leu17]VIP, VIP, NTG, or vehicle. CBF, aortic and ventricular pressures, ventricular contractility (+dp/dtmax) and relaxation (–dp/dtmin) and the EKG were measured. Results: VNS (0.5 ms, 20 V, 5 min.) at 20 Hz maximally increased CBF by 62±14% at 5 min from 71±10 to 115±19 ml/min (p<0.01). VNS at 10, 15, and 30 Hz increased CBF by 6±1%, 24±5%, and 24±7%, respectively (all p<0.05 vs control). Following 20 Hz VNS, CBF returned toward the baseline over 30 min. Aortic and left ventricular (LV) pressures, LV +dp/dtmax and LV –dp/dtmin did not significantly change. After the direct administration of [4Cl-D-Phe6Leu17]VIP into the LCx, VNS increased CBF by only 10±4% (p=NS). Exogenous VIP, in doses of 9.0x10–11 to 2.1x10–9 mol, increased CBF by 106±17% to 169±17% (all p<0.01 vs control). NTG, in doses of 2.2x10–8 to 1.7x10–7 mol, increased CBF by 101±15% to 169±20% (all p<0.01 vs control). These increases in CBF persisted during the 1 to 2 min injection period and returned to the baseline within 5 min. Neither VIP nor NTG significantly changed the heart rate, aortic or LV pressures, LV +dp/dtmax or LV –dp/dtmin. VNS at 20 Hz, exogenous VIP, 9.0x10–11 mol, and exogenous NTG, 2.2x10–8 to 4.4x10–8 mol, produced equivalent increases in CBF by analysis of variance determination. Conclusion: The present experiments suggest that VNS releases VIP which directly dilates coronary arteries and significantly increases coronary artery blood flow.

I really wish I understood these - I'm sure they are quite interesting and informative. Unfortunately I don't have a scientic or medical background - it must really help when dealing with your doctors.

http://www.ebmonline.org/cgi/content/full/227/4/238

Adenosine and Coronary Vasodilation during Ischemia.
Although studies indicate that adenosine is not responsible for functional exercise hyperemia under normal physiological conditions, adenosine most likely contributes significantly to coronary vasodilation during exercise-induced ischemia (34). Studies in chronically instrumented dogs with a coronary stenosis (coronary perfusion pressure = 40 mm Hg) found that coronary vasodilation in response to exercise was significantly reduced by combined blockade of endogenous adenosine production with adenosine deaminase and adenosine receptors with 8-phenyltheophylline (34, 35). These findings indicate that cardiac adenosine release occurs during ischemia (5, 36–38). The critical oxygen tension for adenosine release in isolated cardiomyocytes was estimated to be 3 mm Hg (37). Therefore, as long as changes in myocardial oxygen delivery adequately match changes in the rate of myocardial metabolism, adenosine release is not elevated and thus does not contribute to functional coronary hyperemia. However, if an imbalance between oxygen delivery and consumption is significant, such as in patients with a critical coronary stenosis, adenosine is released by the ischemic myocytes in an attempt to augment coronary blood flow and oxygen delivery.

Potassium Channels
There is a class of potassium channels that can be regulated by intracellular ATP that are present in many tissues, including pancreatic ß cells, skeletal muscle, brain, and smooth muscle (42). These channels are commonly identified by their response to the blocking agent glibenclamide. Glibenclamide is a member of the sulfonylurea class of compounds that have a high selectivity for K+ATP channels, and it has been shown to have a Ki for the channels found in cardiac and smooth muscle in the range of 5–20 µM (48–50). These ATP-sensitive potassium (K+ATP) channels were identified in smooth muscle from mesenteric arteries by Standen et al. in 1989 (44). In 1992, Miyoshi et al. (45) identified K+ATP channels in cultured smooth muscle cells from porcine coronary arteries. Since that time, the role of these channels in the regulation of vessel diameter, particularly in coronary vessels, has been examined by numerous investigators........

....In summary, there is strong evidence that adenosine causes coronary vasodilation during ischemia or hypoxia and that K+ATP channels mediate the vasodilation. Although K+ATP channels are involved in regulating coronary vessel diameter at rest, they are not required during coronary autoregulation or for the increase in coronary blood flow that occurs during exercise.

Nitric Oxide
The coronary vascular endothelium modulates vascular resistance through the production of various vasoactive agents, including the endothelium-derived relaxing factor nitric oxide (63). Nitric oxide is formed from L-arginine by nitric oxide synthase and is continuously released by endothelial cells (64). Nitric oxide release is augmented by agonists such as acetylcholine and bradykinin (64–66) and also by mechanical stimulation such as shear stress (67–73), pulsatile flow (74), and axial strain (75).Endothelial-derived nitric oxide is thought to be one of the factors controlling coronary blood flow both at rest and during exercise. However, inhibition of nitric oxide synthesis with arginine analogues results in either no change (39, 74, 76–85) or a small decrease (86–90) in coronary blood flow at rest and when myocardial oxygen consumption is elevated. Studies also consistently show that blockade of nitric oxide synthesis does not attenuate exercise-induced coronary vasodilation, but does decrease coronary venous oxygen tension at a given level of myocardial oxygen consumption. ......

I'm looking into the connections between the following:

Methylation Cycle
Nitric Oxide Cycle
Acetylcholine
Adenosine
Vagus Nerve
Vasoactive Intestinal Peptide
Coronary blood flow


If the heart is not receiving enough coronary blood flow then perhaps the vagus nerve is stimulated (whether directly in order to compensate for decrease in coronary blood flow or indirectly as a side effect of something else trying to compensate for a decrease in coronary blood flow.) However, due to the great need for oxygenated blood, then the stimulation gets to a degree that becomes adverse and causes orthostatic intolerance.
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http://en.wikipedia.org/wiki/Vagus_nerve
"Parasympathetic innervation of the heart is mediated by the vagus nerve. The right vagus innervates the sinoatrial node. Parasympathetic hyperstimulation predisposes those affected to bradyarrhythmias. The left vagus when hyperstimulated predisposes the heart to atrioventricular (AV) blocks.

At this location Otto Loewi first proved that nerves secrete substances called neurotransmitters which have effects on receptors in target tissues. Loewi described the substance released by the vagus nerve as vagusstoff, which was later found to be acetylcholine.

The vagus nerve has three associated nuclei, the dorsal motor nucleus, the nucleus ambiguus and the solitary nucleus.

Drugs that inhibit the muscarinic cholinergic receptor (anticholinergics) such as atropine and scopolamine are called vagolytic because they inhibit the action of the vagus nerve on the heart, gastrointestinal tract and other organs. Anticholinergic drugs increase heart rate and are used to treat bradycardia (slow heart rate) and asystole, which is when the heart has no electrical activity. Anticholinergic drugs relax the detrusor muscle and cause constipation which again involves the vagus nerve.

Bulimics and anorexics have high vagal activity which is associated with the arrhythmias seen in these patients."

quote:

Homocysteine (a putative cardiovascular risk factor) mounts an oxidative attack on DDAH to form a mixed disulfide, inactivating the enzyme.[5] By oxidizing a sulfhydryl moiety critical for DDAH activity, homocysteine and other risk factors cause ADMA to accumulate and to suppress nitric oxide synthase (NOS) activity.

I happen to know I have high homocysteine. Well, not only do I know it causes a breakdown in connective tissue (including vessels) which causes atherosclerosis---
I now know that homocysteine indirectly suppresses nitric oxide synthase activity.


Here are the Nitric Oxide Synthases and their actions that would be "supressed":

nNOS
Neuronal NOS (nNOS) produces NO in nervous tissue in both the central and peripheral nervous system. Neuronal NOS also performs a role in cell communication and is associated with plasma membranes. nNOS action can be inhibited by NPA (N-propyl-L-arginine). This form of the enzyme is specifically inhibited by 7-nitroindazole.[5]


iNOS
Inducible NOS (iNOS) can be found in the immune system but is also found in the cardiovascular system. It uses the oxidative stress of NO (a free radical) to be used by macrophages in immune defence against pathogens.


eNOS
Main article: Endothelial NOS
Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in blood vessels and is involved with regulating vascular function. A constitutive Ca2+ dependent NOS provides a basal release of NO. eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells.

Sushila sent this to me:

You don't have Martin Pall's book, "Explaining unexplained Illnesses," do you? I wish you did because it is full of material totally relevant to this discussion/ research.

I guess one thing we don't know is your nitric oxide levels.This could be relevant in the high homocysteine..

I'll quote one quick passage from Pall:

"The mechanism of the nitric oxide synthases is verry complex, involving several different cofactors, one of which is tetrahydrobiopterin (BH4). It has been recently shown that when nitric oxide synthases have limited BH4 and limited argenine, they produce superoxide in place of nitric oxide. Because superoxide is another component of the NO/ONOO cycle mechanism, rather than improving these illnesses because of the lowered nitrix oxide production, it is possible that arginine limitation and BH4 depletion may exacerbate them. BH4 availability may be a key factor here, because it is oxidized by peroxynitrite....

It follows from this that the shift towards superoxide production, in addition to the nitric oxide production, essentially converts the three nitric oxide synthases into perozynitrite....(peroxynitrite has sevral actions that deplete energy in the form of ATP...One important response that many cell types have when exposed to high levels of peroxynitrite is apoptosis, programmed cell death....)

the shift of NOS activity towards more superoxide production and nitric oxide production has been called uncoupling, where its intrinsic activity is less tightly coupled to nitric oxide synthesis."

This is me again, BH4 is discussed a lot on the CFS sites. It seems most people are lacking it, some are supplementing it, Amy Yasko says you should not try to raise it too soon...

Maybe you ought to tackle Rich again on Immune Support.

Both Dr. Stewart and Vandy have been investigating Nitric oxide's role in all of this. Have you seen any of that, NiteKitty? I will post when I'm feeling a bit better.

Also, did you know that B12 is an NO scavenger? This would have a similar effect as homocystine - if it is an NO synthase inhibitor..

Reposting this for all who didn't see it before. Has a lot of great info, particularly for those interested in the science and nitty gritty of getting to bottom of it all.