Gaseous signaling molecules

Gaseous signaling molecules are gaseous molecules that are either synthesised internally (endogenously) in the organism, tissue or cell or are received by the organism, tissue or cell from outside (say, from the atmosphere or hydrosphere, as in the case of oxygen) and that are used to transmit chemical signals which induce certain physiological or biochemical changes in the organism, tissue or cell. The term is applied to, for example, oxygen, carbon dioxide, nitric oxide, carbon monoxide, hydrogen sulfide, sulfur dioxide, nitrous oxide, hydrogen cyanide, ammonia, methane, hydrogen, ethylene etc.

Many, but not all, gaseous signaling molecules are called gasotransmitters.

The biological roles of each of the gaseous signaling molecules are in short outlined below.

Gaseous signaling molecules as gasotransmitters

Gasotransmitters is a subfamily of endogenous molecules of gases or gaseous signaling molecules, including NO, CO, H₂S. These particular gases share many common features in their production and function but carry on their tasks in unique ways, which differ from classical signaling molecules, in the human body. In 1981, it was first suggested from clinical work with nitrous oxide that a gas had a direct action at pharmacological receptors and thereby acted as a neurotransmitter. In vitro experiments confirmed these observations which were replicated at NIDA later.

The terminology and characterization criteria of “gasotransmitter” were firstly introduced in 2002. For one gas molecule to be categorized as a gasotransmitters, all of the following criteria should be met.

1. It is a small molecule of gas;
2. It is freely permeable to membranes. As such, its effects do not rely on the cognate membrane receptors. It can have endocrine, paracrine, and autocrine effects. In their endocrine mode of action, for example, gasotransmitters can enter the blood stream; be carried to remote targets by scavengers and released there, and modulate functions of remote target cells;
3. It is endogenously and enzymatically generated and its production is regulated;
4. It has well defined and specific functions at physiologically relevant concentrations. Thus, manipulating the endogenous levels of this gas evokes specific physiological changes;
5. Functions of this endogenous gas can be mimicked by its exogenously applied counterpart;
6. Its cellular effects may or may not be mediated by second messengers, but should have specific cellular and molecular targets.

In 2011, a European Network on Gasotransmitters (ENOG) was formed. The aim of the network is to promote research on NO, CO and H2S in order to better understand the biology of gasotransmitters and to unravel the role of each mediator in health and disease. Moreover, the network aims to contribute to the translation of basic science knowledge in this area of research into therapeutic or diagnostic tools.

Carbon dioxide

Carbon dioxide is one of the mediators of local autoregulation of blood supply. If its levels are high, the capillaries expand to allow a greater blood flow to that tissue.

Bicarbonate ions are crucial for regulating blood pH. A person's breathing rate influences the level of CO₂ in their blood. Breathing that is too slow or shallow causes respiratory acidosis, while breathing that is too rapid leads to hyperventilation, which can cause respiratory alkalosis.

Although the body requires oxygen for metabolism, low oxygen levels normally do not stimulate breathing. Rather, breathing is stimulated by higher carbon dioxide levels.

The respiratory centers try to maintain an arterial CO₂ pressure of 40 mm Hg. With intentional hyperventilation, the CO₂ content of arterial blood may be lowered to 10–20 mm Hg (the oxygen content of the blood is little affected), and the respiratory drive is diminished. This is why one can hold one's breath longer after hyperventilating than without hyperventilating. This carries the risk that unconsciousness may result before the need to breathe becomes overwhelming, which is why hyperventilation is particularly dangerous before free diving.

Nitric oxide

Main article: Biological functions of nitric oxide

NO is one of the few gaseous signalling molecules known and is additionally exceptional due to the fact that it is a radical gas. It is a key vertebrate biological messenger, playing a role in a variety of biological processes. It is a known bioproduct in almost all types of organisms, ranging from bacteria to plants, fungi, and animal cells.

Nitric oxide, known as the 'endothelium-derived relaxing factor', or 'EDRF', is biosynthesized endogenously from L-arginine, oxygen, and NADPH by various nitric oxide synthase (NOS) enzymes. Reduction of inorganic nitrate may also serve to make nitric oxide. The endothelium (inner lining) of blood vessels uses nitric oxide to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. Nitric oxide is highly reactive (having a lifetime of a few seconds), yet diffuses freely across membranes. These attributes make nitric oxide ideal for a transient paracrine (between adjacent cells) and autocrine (within a single cell) signaling molecule.

Independent of nitric oxide synthase, an alternative pathway, coined the nitrate-nitrite-nitric oxide pathway, elevates nitric oxide through the sequential reduction of dietary nitrate derived from plant-based foods. Nitrate-rich vegetables, in particular leafy greens, such as spinach and arugula, and beetroot, have been shown to increase cardioprotective levels of nitric oxide with a corresponding reduction in blood pressure in pre-hypertensive persons. For the body to generate nitric oxide through the nitrate-nitrite-nitric oxide pathway, the reduction of nitrate to nitrite occurs in the mouth, by commensal bacteria, an obligatory and necessary step. Monitoring nitric oxide status by saliva testing detects the bioconversion of plant-derived nitrate into nitric oxide. A rise in salivary levels is indicative of diets rich in leafy vegetables which are often abundant in anti-hypertensive diets such as the DASH diet.

The production of nitric oxide is elevated in populations living at high altitudes, which helps these people avoid hypoxia by aiding in pulmonary vasculature vasodilation. Effects include vasodilatation, neurotransmission (see gasotransmitters), modulation of the hair cycle, production of reactive nitrogen intermediates and penile erections (through its ability to vasodilate). Nitroglycerin and amyl nitrite serve as vasodilators because they are converted to nitric oxide in the body. The vasodilating antihypertensive drug minoxidil contains an NO moiety and may act as an NO agonist. Likewise, Sildenafil citrate, popularly known by the trade name Viagra, stimulates erections primarily by enhancing signaling through the nitric oxide pathway in the penis.

Nitric oxide (NO) contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Humans with atherosclerosis, diabetes, or hypertension often show impaired NO pathways. A high salt intake was demonstrated to attenuate NO production in patients with essential hypertension, although bioavailability remains unregulated.

Nitric oxide is also generated by phagocytes (monocytes, macrophages, and neutrophils) as part of the human immune response. Phagocytes are armed with inducible nitric oxide synthase (iNOS), which is activated by interferon-gamma (IFN-γ) as a single signal or by tumor necrosis factor (TNF) along with a second signal. On the other hand, transforming growth factor-beta (TGF-β) provides a strong inhibitory signal to iNOS, whereas interleukin-4 (IL-4) and IL-10 provide weak inhibitory signals. In this way, the immune system may regulate the armamentarium of phagocytes that play a role in inflammation and immune responses. Nitric oxide is secreted as free radicals in an immune response and is toxic to bacteria and intracellular parasites, including Leishmania and malaria; the mechanism for this includes DNA damage and degradation of iron sulfur centers into iron ions and iron-nitrosyl compounds.

In response, many bacterial pathogens have evolved mechanisms for nitric oxide resistance. Because nitric oxide might have proinflammatory actions in conditions like asthma, there has been increasing interest in the use of exhaled nitric oxide as a breath test in diseases with airway inflammation. Reduced levels of exhaled NO have been associated with exposure to air pollution in cyclists and smokers, but, in general, increased levels of exhaled NO are associated with exposure to air pollution.

Nitric oxide can contribute to reperfusion injury when an excessive amount produced during reperfusion (following a period of ischemia) reacts with superoxide to produce the damaging oxidant peroxynitrite. In contrast, inhaled nitric oxide has been shown to help survival and recovery from paraquat poisoning, which produces lung tissue-damaging superoxide and hinders NOS metabolism.

In plants, nitric oxide can be produced by any of four routes: (i) L-arginine-dependent nitric oxide synthase, (although the existence of animal NOS homologs in plants is debated), (ii) plasma membrane-bound nitrate reductase, (iii) mitochondrial electron transport chain, or (iv) non-enzymatic reactions. It is a signaling molecule, acts mainly against oxidative stress and also plays a role in plant pathogen interactions. Treating cut flowers and other plants with nitric oxide has been shown to lengthen the time before wilting.

Two important biological reaction mechanisms of nitric oxide are S-nitrosation of thiols, and nitrosylation of transition metal ions. S-nitrosation involves the (reversible) conversion of thiol groups, including cysteine residues in proteins, to form S-nitrosothiols (RSNOs). S-Nitrosation is a mechanism for dynamic, post-translational regulation of most or all major classes of protein. The second mechanism, nitrosylation, involves the binding of NO to a transition metal ion like iron or copper. In this function, NO is referred to as a nitrosyl ligand. Typical cases involve the nitrosylation of heme proteins like cytochromes, thereby disabling the normal enzymatic activity of the enzyme. Nitrosylated ferrous iron is particularly stable, as the binding of the nitrosyl ligand to ferrous iron (Fe(II)) is very strong. Hemoglobin is a prominent example of a heme protein that may be modified by NO by both pathways: NO may attach directly to the heme in the nitrosylation reaction, and independently form S-nitrosothiols by S-nitrosation of the thiol moieties.

There are several mechanisms by which NO has been demonstrated to affect the biology of living cells. These include oxidation of iron-containing proteins such as ribonucleotide reductase and aconitase, activation of the soluble guanylate cyclase, ADP ribosylation of proteins, protein sulfhydryl group nitrosylation, and iron regulatory factor activation. NO has been demonstrated to activate NF-κB in peripheral blood mononuclear cells, an important transcription factor in iNOS gene expression in response to inflammation.

It was found that NO acts through the stimulation of the soluble guanylate cyclase, which is a heterodimeric enzyme with subsequent formation of cyclic-GMP. Cyclic-GMP activates protein kinase G, which causes reuptake of Ca²⁺ and the opening of calcium-activated potassium channels. The fall in concentration of Ca²⁺ ensures that the myosin light-chain kinase (MLCK) can no longer phosphorylate the myosin molecule, thereby stopping the crossbridge cycle and leading to relaxation of the smooth muscle cell.

Nitrous oxide

Nitrous oxide in biological systems can be formed by an enzymatic or non-enzymatic reduction of nitric oxide. In vitro studies have shown that endogenous nitrous oxide can be formed by the reaction between nitric oxide and thiol. Some authors have shown that this process of NO reduction to N₂O takes place in hepatocytes, specifically in their cytoplasm and mitochondria, and suggested that the N₂O can possibly be produced in mammalian cells. It is well known that N₂O is produced by some bacteria during process called denitrification.

Apart from its direct and indirect actions at opioid recepters, it was also shown that N₂O inhibits NMDA receptor-mediated activity and ionic currents and diminishes NMDA receptor-mediated excitotoxicity and neurodegeneration. Nitrous oxide also inhibits methionine synthase and slows the conversion of homocysteine to methionine, increases homocysteine concentration and decreases methionine concentration. This effect was shown in lymphocyte cell cultures and in human liver biopsy samples.

Nitrous oxide does not bind as a ligand to the heme and does not react with thiol-containing proteins. Nevertheless, studies have shown that nitrous oxide can reversibly and non-covalently "insert" itself into the inner structures of some heme-containing proteins such as hemoglobin, myoglobin, cytochrome oxidase and alter their structure and function. The ability of nitrous oxide to alter the structure and function of these proteins was demonstrated by shifts in infrared spectra of cysteine thiols of hemoglobin and by partial and reversible inhibition of cytochrome oxidase.

Endogenous nitrous oxide can possibly play a role in modulating endogenous opioid and NMDA systems.

Carbon monoxide

Main article: Biological functions of carbon monoxide

Carbon monoxide is produced naturally by the human body as a signaling molecule. Thus, carbon monoxide may have a physiological role in the body, such as a neurotransmitter or a blood vessel relaxant. Because of carbon monoxide's role in the body, abnormalities in its metabolism have been linked to a variety of diseases, including neurodegenerations, hypertension, heart failure, and inflammation.

Functional Summary

• CO functions as an endogenous signaling molecule.
• CO modulates functions of the cardiovascular system.
• CO inhibits blood platelet aggregation and adhesion.
• CO may play a role as potential therapeutic agent.

In mammals, carbon monoxide is naturally produced by the action of heme oxygenase 1 and 2 on the heme from hemoglobin breakdown. This process produces a certain amount of carboxyhemoglobin in normal persons, even if they do not breathe any carbon monoxide.

Following the first report that carbon monoxide is a normal neurotransmitter in 1993, as well as one of three gases that naturally modulate inflammatory responses in the body (the other two being nitric oxide and hydrogen sulfide), carbon monoxide has received a great deal of clinical attention as a biological regulator. In many tissues, all three gases are known to act as anti-inflammatories, vasodilators, and encouragers of neovascular growth. However, the issues are complex, as neovascular growth is not always beneficial, since it plays a role in tumor growth, and also the damage from wet macular degeneration, a disease for which smoking (a major source of carbon monoxide in the blood, several times more than natural production) increases the risk from 4 to 6 times.

There is a theory that, in some nerve cell synapses, when long-term memories are being laid down, the receiving cell makes carbon monoxide, which back-transmits to the transmitting cell, telling it to transmit more readily in future. Some such nerve cells have been shown to contain guanylate cyclase, an enzyme that is activated by carbon monoxide.

Studies involving carbon monoxide have been conducted in many laboratories throughout the world for its anti-inflammatory and cytoprotective properties. These properties have potential to be used to prevent the development of a series of pathological conditions including ischemia reperfusion injury, transplant rejection, atherosclerosis, severe sepsis, severe malaria, or autoimmunity. Clinical tests involving humans have been performed, however the results have not yet been released.

Carbon suboxide

Those are 6- or 8-ring macrocyclic polymers of carbon suboxide that were found in living organisms. They are acting as an endogenous digoxin-like Na+/K+-ATP-ase and Ca-dependent ATP-ase inhibitors, endogenous natriuretics, antioxidants and antihypertensives

Carbon suboxide, C₃O₂, can be produced in small amounts in any biochemical process that normally produces carbon monoxide, CO, for example, during heme oxidation by heme oxygenase-1. It can also be formed from malonic acid. It has been shown that carbon suboxide in an organism can quickly polymerize into macrocyclic polycarbon structures with the common formula (C₃O₂)_n (mostly (C₃O₂)₆ and (C₃O₂)₈), and that those macrocyclic compounds are potent inhibitors of Na⁺/K⁺-ATP-ase and Ca-dependent ATP-ase, and have digoxin-like physiological properties and natriuretic and antihypertensive actions. Those macrocyclic carbon suboxide polymer compounds are thought to be endogenous digoxin-like regulators of Na⁺/K⁺-ATP-ases and Ca-dependent ATP-ases, and endogenous natriuretics and antihypertensives. Other than that, some authors think also that those macrocyclic compounds of carbon suboxide can possibly diminish free radical formation and oxidative stress and play a role in endogenous anticancer protective mechanisms, for example in the retina.

Hydrogen sulfide

Main article: Biological functions of hydrogen sulfide

Hydrogen sulfide is produced in small amounts by some cells of the mammalian body and has a number of biological signaling functions. (Only two other such gases are currently known: nitric oxide (NO) and carbon monoxide (CO).)

The gas is produced from cysteine by the enzymes cystathionine beta-synthase and cystathionine gamma-lyase. It acts as a relaxant of smooth muscle and as a vasodilator and is also active in the brain, where it increases the response of the NMDA receptor and facilitates long term potentiation, which is involved in the formation of memory.

Eventually the gas is converted to sulfite in the mitochondria by thiosulfate reductase, and the sulfite is further oxidized to thiosulfate and sulfate by sulfite oxidase. The sulfates are excreted in the urine.

Due to its effects similar to nitric oxide (without its potential to form peroxides by interacting with superoxide), hydrogen sulfide is now recognized as potentially protecting against cardiovascular disease. The cardioprotective role effect of garlic is caused by catabolism of the polysulfide group in allicin to H
₂S, a reaction that could depend on reduction mediated by glutathione.

Though both nitric oxide (NO) and hydrogen sulfide have been shown to relax blood vessels, their mechanisms of action are different: while NO activates the enzyme guanylyl cyclase, H
₂S activates ATP-sensitive potassium channels in smooth muscle cells. Researchers are not clear how the vessel-relaxing responsibilities are shared between nitric oxide and hydrogen sulfide. However, there exists some evidence to suggest that nitric oxide does most of the vessel-relaxing work in large vessels and hydrogen sulfide is responsible for similar action in smaller blood vessels.

Recent findings suggest strong cellular crosstalk of NO and H
₂S, demonstrating that the vasodilatatory effects of these two gases are mutually dependent. Additionally, H
₂S reacts with intracellular S-nitrosothiols to form the smallest S-nitrosothiol (HSNO), and a role of hydrogen sulfide in controlling the intracellular S-nitrosothiol pool has been suggested.

Like nitric oxide, hydrogen sulfide is involved in the relaxation of smooth muscle that causes erection of the penis, presenting possible new therapy opportunities for erectile dysfunction.

Hydrogen sulfide (H2S) deficiency can be detrimental to the vascular function after an acute myocardial infarction (AMI). AMIs can lead to cardiac dysfunction through two distinct changes; increased oxidative stress via free radical accumulation and decreased NO bioavailability. Free radical accumulation occurs due to increased electron transport uncoupling at the active site of endothelial nitric oxide synthase (eNOS), an enzyme involved in converting L-arginine to NO. During an AMI, oxidative degradation of tetrahydrobiopterin (BH4), a cofactor in NO production, limits BH4 availability and limits NO productionby eNOS. Instead, eNOS reacts with oxygen, another cosubstrates involved in NO production. The products of eNOS are reduced to superoxides, increasing free radical production and oxidative stress within the cells. A H2S deficiency impairs eNOS activity by limiting Akt activation and inhibiting Akt phosphorylation of the eNOSS1177 activation site. Instead, Akt activity is increased to phosphorylate the eNOST495 inhibition site, downregulating eNOS production of NO.

H2S therapy uses a H2S donor, such as diallyl trisulfide (DATS), to increase the supply of H2S to an AMI patient. H2S donors reduce myocardial injury and reperfusion complications. Increased H2S levels within the body will react with oxygen to produce sulfane sulfur, a storage intermediate for H2S. H2S pools in the body attracts oxygen to react with excess H2S and eNOS to increase NO production. With increased use of oxygen to produce more NO, less oxygen is available to react with eNOS to produce superoxides during an AMI, ultimately lowering the accumulation of reactive oxygen species (ROS). Furthermore, decreased accumulation of ROS lowers oxidative stress in vascular smooth muscle cells, decreasing oxidative degeneration of BH4. Increased BH4 cofactor contributes to increased production of NO within the body. Higher concentrations of H2S directly increase eNOS activity through Akt activation to increase phosphorylation of the eNOSS1177 activation site, and decrease phosphorylation of the eNOST495 inhibition site. This phosphorylation process upregulates eNOS activity, catalyzing more conversion of L-arginine to NO. Increased NO production enables soluble guanylyl cyclase (sGC) activity, leading to an increased conversion of guanosine triphosphate (GTP) to 3’,5’-cyclic guanosine monophosphate (cGMP). In H2S therapy immediately following an AMI, increased cGMP triggers an increase in protein kinase G (PKG) activity. PKG reduces intracellular Ca2+ in vascular smooth muscle to increase smooth muscle relaxation and promote blood flow. PKG also limits smooth muscle cell proliferation, reducing intima thickening following AMI injury, ultimately decreasing myocardial infarct size.

In Alzheimer's disease the brain's hydrogen sulfide concentration is severely decreased. In a certain rat model of Parkinson's disease, the brain's hydrogen sulfide concentration was found to be reduced, and administering hydrogen sulfide alleviated the condition. In trisomy 21 (Down syndrome) the body produces an excess of hydrogen sulfide. Hydrogen sulfide is also involved in the disease process of type 1 diabetes. The beta cells of the pancreas in type 1 diabetes produce an excess of the gas, leading to the death of these cells and to a reduced production of insulin by those that remain.

In 2005, it was shown that mice can be put into a state of suspended animation-like hypothermia by applying a low dosage of hydrogen sulfide (81 ppm H
₂S) in the air. The breathing rate of the animals sank from 120 to 10 breaths per minute and their temperature fell from 37 °C to just 2 °C above ambient temperature (in effect, they had become cold-blooded). The mice survived this procedure for 6 hours and afterwards showed no negative health consequences. In 2006 it was shown that the blood pressure of mice treated in this fashion with hydrogen sulfide did not significantly decrease.

A similar process known as hibernation occurs naturally in many mammals and also in toads, but not in mice. (Mice can fall into a state called clinical torpor when food shortage occurs). If the H
₂S-induced hibernation can be made to work in humans, it could be useful in the emergency management of severely injured patients, and in the conservation of donated organs. In 2008, hypothermia induced by hydrogen sulfide for 48 hours was shown to reduce the extent of brain damage caused by experimental stroke in rats.

As mentioned above, hydrogen sulfide binds to cytochrome oxidase and thereby prevents oxygen from binding, which leads to the dramatic slowdown of metabolism. Animals and humans naturally produce some hydrogen sulfide in their body; researchers have proposed that the gas is used to regulate metabolic activity and body temperature, which would explain the above findings.

Two recent studies cast doubt that the effect can be achieved in larger mammals. A 2008 study failed to reproduce the effect in pigs, concluding that the effects seen in mice were not present in larger mammals. Likewise a paper by Haouzi et al. noted that there is no induction of hypometabolism in sheep, either.

At the February 2010 TED conference, Mark Roth announced that hydrogen sulfide induced hypothermia in humans had completed Phase I clinical trials. The clinical trials commissioned by the company he helped found, Ikaria, were however withdrawn or terminated by August 2011.

Sulfur dioxide

The role of sulfur dioxide in mammalian biology is not yet well understood. Sulfur dioxide blocks nerve signals from the pulmonary stretch receptors and abolishes the Hering–Breuer inflation reflex.

It was shown that endogenous sulfur dioxide plays a role in diminishing an experimental lung damage caused by oleic acid. Endogenous sulfur dioxide lowered lipid peroxidation, free radical formation, oxidative stress and inflammation during an experimental lung damage. Conversely, a successful lung damage caused a significant lowering of endogenous sulfur dioxide production, and an increase in lipid peroxidation, free radical formation, oxidative stress and inflammation. Moreover, blockade of an enzyme that produces endogenous SO₂ significantly increased the amount of lung tissue damage in the experiment. Conversely, adding acetylcysteine or glutathione to the rat diet increased the amount of endogenous SO₂ produced and decreased the lung damage, the free radical formation, oxidative stress, inflammation and apoptosis.

It is considered that endogenous sulfur dioxide plays a significant physiological role in regulating cardiac and blood vessel function, and aberrant or deficient sulfur dioxide metabolism can contribute to several different cardiovascular diseases, such as arterial hypertension, atherosclerosis, pulmonary arterial hypertension, stenocardia.

It was shown that in children with pulmonary arterial hypertension due to congenital heart diseases the level of homocysteine is higher and the level of endogenous sulfur dioxide is lower than in normal control children. Moreover, these biochemical parameters strongly correlated to the severity of pulmonary arterial hypertension. Authors considered homocysteine to be one of useful biochemical markers of disease severity and sulfur dioxide metabolism to be one of potential therapeutic targets in those patients.

Endogenous sulfur dioxide also has been shown to lower the proliferation rate of endothelial smooth muscle cells in blood vessels, via lowering the MAPK activity and activating adenylyl cyclase and protein kinase A. Smooth muscle cell proliferation is one of important mechanisms of hypertensive remodeling of blood vessels and their stenosis, so it is an important pathogenetic mechanism in arterial hypertension and atherosclerosis.

Endogenous sulfur dioxide in low concentrations causes endothelium-dependent vasodilation. In higher concentrations it causes endothelium-independent vasodilation and has a negative inotropic effect on cardiac output function, thus effectively lowering blood pressure and myocardial oxygen consumption. The vasodilating effects of sulfur dioxide are mediated via ATP-dependent calcium channels and L-type ("dihydropyridine") calcium channels. Endogenous sulfur dioxide is also a potent antiinflammatory, antioxidant and cytoprotective agent. It lowers blood pressure and slows hypertensive remodeling of blood vessels, especially thickening of their intima. It also regulates lipid metabolism.

Endogenous sulfur dioxide also diminishes myocardial damage, caused by isoproterenol adrenergic hyperstimulation, and strengthens the myocardial antioxidant defense reserve.

Sulfur dioxide expelled from the gastrointestinal tract (through the process of flatulence) is thought to be one of the main constituents to the unpleasant smell colloquially referred to as a "fart".

Hydrogen cyanide

Some authors have shown that neurons can produce hydrogen cyanide upon activation of their opioid receptors by endogenous or exogenous opioids. They have also shown that neuronal production of HCN activates NMDA receptors and plays a role in signal transduction between neuronal cells (neurotransmission). Moreover, increased endogenous neuronal HCN production under opioids was seemingly needed for adequate opioid analgesia, as analgesic action of opioids was attenuated by HCN scavengers. They considered endogenous HCN to be a neuromodulator.

It was also shown that, while stimulating muscarinic cholinergic receptors in cultured pheochromocytoma cells increases HCN production, in a living organism (in vivo) muscarinic cholinergic stimulation actually decreases HCN production.

Leukocytes generate HCN during phagocytosis.

The vasodilatation, caused by sodium nitroprusside, has been shown to be mediated not only by NO generation, but also by endogenous cyanide generation, which adds not only toxicity, but also some additional antihypertensive efficacy compared to nitroglycerine and other non-cyanogenic nitrates which do not cause blood cyanide levels to rise.

Ammonia

Ammonia also plays a role in both normal and abnormal animal physiology. It is biosynthesised through normal amino acid metabolism and is toxic in high concentrations. The liver converts ammonia to urea through a series of reactions known as the urea cycle. Liver dysfunction, such as that seen in cirrhosis, may lead to elevated amounts of ammonia in the blood (hyperammonemia). Likewise, defects in the enzymes responsible for the urea cycle, such as ornithine transcarbamylase, lead to hyperammonemia. Hyperammonemia contributes to the confusion and coma of hepatic encephalopathy, as well as the neurologic disease common in people with urea cycle defects and organic acidurias.

Ammonia is important for normal animal acid/base balance. After formation of ammonium from glutamine, α-ketoglutarate may be degraded to produce two molecules of bicarbonate, which are then available as buffers for dietary acids. Ammonium is excreted in the urine, resulting in net acid loss. Ammonia may itself diffuse across the renal tubules, combine with a hydrogen ion, and thus allow for further acid excretion.

Methane

Some authors have shown that endogenous methane is produced not only by the intestinal flora and then absorbed into the blood, but also is produced - in small amounts - by eukaryotic cells (during process of lipid peroxidation). And they have also shown that the endogenous methane production rises during an experimental mitochondrial hypoxia, for example, sodium azide intoxication. They thought that methane could be one of intercellular signals of hypoxia and stress.

Other authors have shown that cellular methane production also rises during sepsis or bacterial endotoxemia, including an experimental imitation of endotoxemia by lipopolysaccharide (LPS) administration.

Some other researchers have shown that methane, produced by the intestinal flora, is not fully "biologically neutral" to the intestine, and it participates in the normal physiologic regulation of peristalsis. And its excess causes not only belching, flatulence and belly pain, but also functional constipation.

Ethylene

An ethylene signal transduction pathway. Ethylene permeates the membrane and binds to a receptor on the endoplasmic reticulum. The receptor releases the repressed EIN2. This then activates a signal transduction pathway which activates a regulatory genes that eventually trigger an Ethylene response. The activated DNA is transcribed into mRNA which is then translated into a functional enzyme that is used for ethylene biosynthesis.

Ethylene serves as a hormone in plants. It acts at trace levels throughout the life of the plant by stimulating or regulating the ripening of fruit, the opening of flowers, and the abscission (or shedding) of leaves. Commercial ripening rooms use "catalytic generators" to make ethylene gas from a liquid supply of ethanol. Typically, a gassing level of 500 to 2,000 ppm is used, for 24 to 48 hours. Care must be taken to control carbon dioxide levels in ripening rooms when gassing, as high temperature ripening (20 °C; 68 °F) has been seen to produce CO₂ levels of 10% in 24 hours.

Ethylene has been used since the ancient Egyptians, who would gash figs in order to stimulate ripening (wounding stimulates ethylene production by plant tissues). The ancient Chinese would burn incense in closed rooms to enhance the ripening of pears. In 1864, it was discovered that gas leaks from street lights led to stunting of growth, twisting of plants, and abnormal thickening of stems. In 1901, a Russian scientist named Dimitry Neljubow showed that the active component was ethylene. Sarah Doubt discovered that ethylene stimulated abscission in 1917. It wasn't until 1934 that Gane reported that plants synthesize ethylene. In 1935, Crocker proposed that ethylene was the plant hormone responsible for fruit ripening as well as senescence of vegetative tissues.

The Yang cycle

Ethylene is produced from essentially all parts of higher plants, including leaves, stems, roots, flowers, fruits, tubers, and seeds. Ethylene production is regulated by a variety of developmental and environmental factors. During the life of the plant, ethylene production is induced during certain stages of growth such as germination, ripening of fruits, abscission of leaves, and senescence of flowers. Ethylene production can also be induced by a variety of external aspects such as mechanical wounding, environmental stresses, and certain chemicals including auxin and other regulators.

Ethylene is biosynthesized from the amino acid methionine to S-adenosyl-L-methionine (SAM, also called Adomet) by the enzyme Met Adenosyltransferase. SAM is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase (ACS). The activity of ACS determines the rate of ethylene production, therefore regulation of this enzyme is key for the ethylene biosynthesis. The final step requires oxygen and involves the action of the enzyme ACC-oxidase (ACO), formerly known as the ethylene forming enzyme (EFE). Ethylene biosynthesis can be induced by endogenous or exogenous ethylene. ACC synthesis increases with high levels of auxins, especially indole acetic acid (IAA) and cytokinins.

Ethylene is perceived by a family of five transmembrane protein dimers such as the ETR₁ protein in Arabidopsis. The gene encoding an ethylene receptor has been cloned in Arabidopsis thaliana and then in tomato. Ethylene receptors are encoded by multiple genes in the Arabidopsis and tomato genomes. Mutations in any of the gene family, which comprises five receptors in Arabidopsis and at least six in tomato, can lead to insensitivity to ethylene. DNA sequences for ethylene receptors have also been identified in many other plant species and an ethylene binding protein has even been identified in Cyanobacteria.

Environmental cues such as flooding, drought, chilling, wounding, and pathogen attack can induce ethylene formation in plants. In flooding, roots suffer from lack of oxygen, or anoxia, which leads to the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC). ACC is transported upwards in the plant and then oxidized in leaves. The ethylene produced causes nastic movements (epinasty) of the leaves, perhaps helping the plant to lose water.

Ethylene in plant induces such responses:

• Seedling triple response, thickening and shortening of hypocotyl with pronounced apical hook.
• In pollination, when the pollen reaches the stigma, the precursor of the ethene, ACC, is secreted to the petal, the ACC releases ethylene with ACC oxidase.
• Stimulates leaf and flower senescence
• Stimulates senescence of mature xylem cells in preparation for plant use
• Induces leaf abscission
• Induces seed germination
• Induces root hair growth — increasing the efficiency of water and mineral absorption
• Induces the growth of adventitious roots during flooding
• Stimulates epinasty — leaf petiole grows out, leaf hangs down and curls into itself
• Stimulates fruit ripening
• Induces a climacteric rise in respiration in some fruit which causes a release of additional ethylene.
• Affects gravitropism
• Stimulates nutational bending
• Inhibits stem growth and stimulates stem and cell broadening and lateral branch growth outside of seedling stage (see Hyponastic response)
• Interference with auxin transport (with high auxin concentrations)
• Inhibits shoot growth and stomatal closing except in some water plants or habitually flooded ones such as some rice varieties, where the opposite occurs (conserving CO
  ₂ and O
  ₂)
• Induces flowering in pineapples
• Inhibits short day induced flower initiation in Pharbitus nil and Chrysanthemum morifolium

Small amounts of endogenous ethylene are also produced in mammals, including humans, due to lipid peroxidation. Some of endogenous ethylene is then oxidized to ethylene oxide, which is able to alkylate DNA and proteins, including hemoglobin (forming a specific adduct with its N-terminal valine, N-hydroxyethyl-valine). Endogenous ethylene oxide, just as like environmental (exogenous) one, can alkylate guanine in DNA, forming an adduct 7-(2-hydroxyethyl)-guanine, and this poses an intrinsic carcinogenic risk. It is also mutagenic.