104 South African Journal of Science 103, March/April 2007 Research in Action

between CO2 and N2O in the Nitrous and dioxide: and . their similar and contrasting , like , is produced endogenously biological effects Nitrous oxide is a metabolic product of certain , some of which are † M.A. Gillman*‡ and F.J. Lichtigfeld* commensals in the gut of animals includ- ing man.10 In recent years, moreover, evidence has appeared indicating that

N2O may be produced in mammalian he similar and contrasting biological 4,7 the CO2 involved in . cells as part of their . When properties of CO2 and N2O appear to acquire their either directly we discovered that N2O could be involved Treflect fundamental biological processes from the or through internal recycling in neurotransmission in humans,11–13 we in both very simple and the most complex . of and . The amount of did not realize that we had stumbled on a ammonium recyling in C3 plants is signifi- new biological principle. The generality of our finding was confirmed when nitric Introduction cantly higher than in C4 plants as a result A recent article in this journal1 discussed of photorespiration. As the rate of ammo- oxide (NO), another endogenously pro- duced , was also discovered to have a the similarities and differences between nia production in C3 plants can be 20 times the rate of primary nitrate , role in neurotransmission.14,15 nitrous oxide (N2O) and carbon dioxide the cannot afford to lose this ammo- The evidence that N2O is produced (CO2) from a physico-chemical perspective. 4,7 Here we consider the apparent similarities nia as a volatile gas but must have an endogenously seems to confirm the and differences between these two effective mechanism for its re-assimi- possibility that it is not only the first gas to in a biological context. lation. Three (glutamine synthe- have been shown to have a role in neuro- tase, and glutamate transmission but that it is formed by the It is well known that biologically active 16,17 chemical agents can act directly with ) have been identified in of NO. This once again highlights the similarities of N O and receptors in the brain to produce physio- transforming into amino 2 2 CO , which both appear to be endoge- logical and pharmacological effects. but there is debate as to the role of the 2 These receptors have unique and specific various oxidized nitrogen intermediates nously produced as metabolic products in physico-chemical structures and proper- which are formed.3 It is possible that bacterial and mammalian cells. These ties. Chemical substances that attach to one of these is NO, which may be gases are therefore involved in the physi- ology of cells from the simplest to the these receptors do so by characteristic reduced to N2O as has been shown in physico-chemical mechanisms, which mammalian cells4 and bacteria.5 In sup- most complex forms. This is perhaps not surprising, given that N O and CO then trigger a cascade of events through port of this idea, plants have been shown 2 2 are linked through the nitrogen and the interaction of the particular agent to emit endogenously produced N2O 2 carbon cycles (see above), which are with the specific . It is therefore during nitrate assimilation.6 not surprising that the structural similari- crucial to living organisms. Nitrous oxide does not substitute for O2 ties of the N2O and CO2 translate in either the cytochrome c oxidase or Actions on the into similar biological actions. On the carbon (CO) diogenase reac- In higher animals, N O and CO have other hand, such structural and other tions that produce, respectively, 2 2 related actions on the nervous system. differences as they have may account for and CO and which are both catalysed by 2 Both gases in high concentrations cause their dissimilar biological effects. cytochrome c oxidase.7 Rather, it acts by depression of the , reducing the rate of electron transfer from Nitrogen and carbon cycles leading eventually to , respiratory cytochrome c to the O reaction site.7 It is Photorespiration involves the - 2 depression and finally death.18–20 Despite therefore possible that N O interacts with dependent of CO and uptake 2 these apparent similarities, however, 2 CO in the plant by reducing the flow of in green . During the 2 there are differences in their biological of electrons to both CO and O reaction 19,20 1950s and 1960s, the synthesis of glycolate 2 2 effects. sites. The latter postulate is supported by by leaves in high O concentrations or low 2 the finding that N O inhibits the use of O CO settings was demonstrated and the 2 2 2 by plant .8 enzymic pathways involved were eluci- The effects of N2O on respiratory drive 3 The link between N Oand CO through dated. bisphosphate carboxy- 2 2 are relatively small, with slight depression lase/ (, RuBP) is the the carbon and nitrogen cycles is also seen of the stimulatory response in humans to in the composition of the atmosphere and 19 primary involved, with both CO2 CO2 when 50% N2O in oxygen is breathed. during glacial cycles.9 The end of a and O2 competing for its active site, the The response to hypoxia is also reduced glacial cycle is marked by the position on the enzyme to which the sub- when 50% N2O is breathed. Since N2Oat strate binds. There is a well-established of N2O, a gas, from water. non-anaesthetic concentrations (such as link between photorespiration and the This is described by a nonlinear model 50%) is an multipotent ,21–23 in plants. During the which takes into account the effect of its effect on respiration could of , ammonia is variations in the ocean’s content of fixed be opioid mediated. Nevertheless, it is produced in stoichiometric amounts with nitrogen following enhanced denitrifi- much less marked than some other cation.9 Outgassing to greater levels and particularly those that are selective *South African Brain Research Institute, 6 Campbell St, Waverley, Johannesburg 2090, South Africa. of atmospheric CO2 during interglacial mu-opioid , such as . † 9 Deceased. periods, showing a direct relationship Competitive opioid antagonists such as ‡Author for correspondence. E-mail: [email protected] Research in Action South African Journal of Science 103, March/April 2007 105

nalorphine and naloxone can reverse consequences are almost certainly recep- Other workers have shown that both 24,25 16,17,21,22,28,31–33 these effects on respiration. Such antag- tor mediated and could also be N2O and hyperventilation have similar onistic outcomes on respiratory depression responsible for its actions on the respira- inhibitory effects on lamina V of the spinal are extremely rapid as would be expected tory centres. cord, where there is convergence of visceral because they are mediated by direct effects and high threshold cutaneous pain.40 on opioid receptors. Moreover, endoge- Sympathetic activity These laminae are rich in opioid termi- nous and exogenous opioids, including Sympathetic activity, which prepares nals.41

N2O, inhibit the responses of the brain- the body for the fright/flight response, is These findings seem to indicate that stem respiratory centre to increased CO increased by both N O34,35 and CO .20 2 2 2 CO2 , like N2O, acts directly on mu-opioid 19 levels. Thus, the control of respira- Nitrous oxide seems to act directly at receptors, especially in their effects on tory ‘may be directly related to opioid receptors as well as via the alpha2- pain perception and possibly also in their 36 competition for opioid receptor sites by system. Carbon dioxide actions on respiration as suggested previ- these various gases as well as endogenous potently activates the central adrenergic ously (see above) on the basis of radio- 22 20 opioid agents.’ It is therefore significant system and there is some preliminary receptor assays.22 that the depressant effect of morphine on evidence that it acts directly on mu-opioid 28 respiration can be as rapidly reversed by receptors, further underlining the simi- Other pharmacological actions of CO 26 2 inhalation of 5% CO2 in oxygen, indicat- larity between the biological actions of and N2O on the central nervous system ing the possibility that this effect is also these two gases. The descending adre- Pharmacological, rather than physio- receptor mediated. nergic and nor-adrenergic cell groups logical, concentrations of CO2 produce 42,43 That this influence of CO2 is likely to form most of the lateral medullary stress reactions such as anxiety and fear. involve receptors is supported by the fact reticular formation and are linked to This outcome is probably mediated by that stimulation of ventilation in man various vital centres in the medulla stimulation of the sympathetic system20 begins within seconds of inhaling low oblongata of the brainstem, which includes and blockade of opioid receptors.26 In 37 concentrations of the gas, with a maximum the respiratory centres. contrast, although also heightening sym- response occurring within less than 5 Apart from the influence of these gases 19,20 20 pathetic stimulation, non-anaesthetic minutes. These stimulatory consequences on the adrenergic system, they also have doses of N O have anti-stress effects44–47 of CO inhalation disappear rapidly after 2 2 local effects. One which they share is the where the agonistic influence of N Oon its withdrawal.20 Indeed, the CO effect on direct local influence on systemic blood 2 2 the opioid receptors seems to outweigh its medullary and peripheral 20,38,39 vessels, resulting in vasodilatation. sympathetic activity.45 Indeed, low doses arterial chemoreceptors are clearly recep- The results of central sympathetic activa- 46 of N O reduce cortisol release. tor mediated.20 In these circumstances, it tion are usually opposite to the local effects 2 Both CO and N O have is likely that the effects of CO on the of CO , whereas the local influence 2 2 2 2 effects.48–50 Interestingly, found that ventilatory neurons in the medullary– on blood vessels seems to be more impor- such effects of CO were magnified if pontine region are also mediated by re- tant than are the adrenergically medi- 2 preceded by a short administration of ceptors. A further clue that this is an ated vasoconstrictor effects.20 Nitrous anaesthetic concentrations of N Ofor8or opioid interaction is that, in barbiturate oxide35,36,38 shares these properties. 2 9 inhalations. Nitrous oxide at 100% used poisoning, which is not mediated by 27 as an anaesthetic, also for a few inhala- opioid receptors, the administration of Pain perception tions (less than a minute’s exposure), has CO2 has no positive effect on stimulating Analgesia can be produced by hyper- 49 26 an antidepressant action. Furthermore, respiration. That various gases influence capnia, which triggers the release of inhalation of 80% N O (with 20% O ) for respiration by interacting directly at 2 2 endogenous opioids, presumably from 50 opioid receptors is supported by the find- the spinal dorsal horn,29 a region in the between 1.5 and 5 minutes demon- ing that 100% oxygen altered the in vitro spinal cord involved with pain perception. strated that the gas has antidepressant ef- 3 fects. Low concentrations of N O mixed binding of H-dihydromorphine as well The dorsal horn appears to be implicated 2 3 28 with high concentrations of O titrated to as that of H-ketocyclazocine. Dihydro- because the modification of tail-flick 2 morphine and ketocyclazocine are opioid latency persists after spinalization.29 The a level where the patient is relaxed but receptor , so that evidence that in authors responsible for these results con- fully conscious [so-called psychotropic 17 vitro binding with these agents is inter- cluded that may produce nitrous oxide (PAN)] also have 51 fered with by oxygen indicates that direct excitatory effects on spinal or marked antidepressant properties. 50,51 oxygen itself also binds to opioid recep- bulbo-spinal enkephalinergic (that is, Moreover, this work indicates that tors and therefore has opioid proper- opioid) interneurones. Since this effect endogenous depressions (which are ties. Other studies have confirmed this was also present in animals spinalized at caused by an underlying biological work.31 lower thoracic levels, at least one popula- disturbance) are resistant to the effects of N O, whereas reactive depressions (which Although binding studies with CO2 tion of these neurones must have been in 2 using opioid ligands have not been the lumbo-sacral region.29 Gamble and are related to a stressful life event) are ameliorated by exposure to the gas. performed, as far as we are aware, the Milne draw the analogy with N2O when similarity of the actions of the specific they state that: ‘It is interesting that Carbon dioxide at high concentrations opioid antagonistic, naloxone, on CO2 analgesia induced by the structurally (75% in O2 administered for 30–120 29 and N2O analgesia (see below) may similar compound nitrous oxide can be seconds) is effective for treating addicts indicate that CO2 also binds to opioid partly blocked by relatively high doses of from whom abused substances such as receptors, to exert some of its respiratory naloxone under some circumstances.’ alcohol52,53 and opioids54,55 are suddenly 56 effects. They found that analgesia caused by CO2 withdrawn. PAN has also been used 53,57 The pharmacological effects of N2O are was also blocked with relatively high successfully to treat both acute also rapid in onset and offset19,30 and these doses of naloxone (2 mg/kg).29 and opioid58,59 withdrawal states. 106 South African Journal of Science 103, March/April 2007 Research in Action

Conclusions comments. 367, 28. 37. Williams P.L. (Ed.) (1995). Gray’s , 38th edn, pp. 1076–1077. Churchill Livingstone, New Although we have touched on only 17. Gillman M.A. and Lichtigfeld F.J. (1994). Opioid properties of psychotropic analgesic nitrous oxide York. some of the properties of these gases, the (laughing gas). Perspect. Biol. Med. 38, 125–138. 38. Matheny J.L., Westphal K.A., Richardson D.R. similar but distinct differences in molecular 18. Ganong W.F. (1995). In Review of Medical Physiol- and Roth G.I. (1991). Macro- and microvascular structure of CO and N O seem to be ogy, 17th edn, pp. 625–639. Prentice-Hall, London. effects of nitrous oxide in the rat. Anesth. Prog. 38, 2 2 19. Marshall B.E. and Longnecker D.E. (1996). 57–64. reflected in their similar but contrasting General . In Goodman and Gilman’s The 39. Roth G.I, Matheny J.L., Falace D.A., O’Reilly J.E. biological actions. It is clear that these Pharmacological Basis of Therapeutics, 9th edn, ed. and Norton J.C. (1984). Effect of age on the digit gases have overlapping effects. However, J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. blood flow response to concentrations of Ruddon and A.G. Gilman, pp. 307–330. McGraw- nitrous oxide. Anesth. Prog. 31, 17–22. CO2 stimulates respiration whereas N2O Hill, . 40. Kitahata L.M, Taub A. and Sato I. (1971). Lamina- has a slight antagonistic influence on 20. Eickenhoff R.G. and Longnecker D.E. (1996). The specific suppression of dorsal horn unit activ- therapeutic gases. In Goodman and Gilman’s The ity by nitrous oxide and by hyperventilation. the stimulatory consequences of CO2. Another distinction is that pharmacologi- Pharmacological Basis of Therapeutics, 9th edn, ed. J. Pharmacol. Exp. Ther. 176, 101–108. J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. 41. Gillman M.A. and Lichtigfeld F.J. (1985). A cal concentrations of CO2 produce a stress Ruddon and A.G. Gilman, pp. 349–359. McGraw- pharmacological overview of opioid mechanisms mediating analgesia and hyperalgesia. Neurolog. reaction, while the reverse is true of N2O. Hill, New York Moreover, although both gases can ame- 21. Gillman M.A. (1986). Analgesic (sub-) Res. 7, 106–119. nitrous oxide interacts with the endogenous 42. LaVerne A.A. (1953). Rapid coma technique of liorate withdrawal states after substance opioid system: a review of the evidence. Life Sci. carbon dioxide inhalation therapy. Dis. Nerv. Syst. abuse, the duration of exposure and the 39, l209–l22l. 14, 141–44. concentrations of the gases required are 22. Gillman M.A. (1989). Physico-chemical basis of 43. Scott W., M.D., Chrystal J.H., Cynthia L., different. nitrous oxide activity at opioid receptors. S. Afr. J. D’Amico C.L., Heninger M.D. and Charney M.D. Sci. 85, 280–281. (1988). A review of behavioural and pharmaco-

We thank Mary Scholes and Mike Cramer for advice 23. Gillman M.A. (1993). Analgesic nitrous oxide logic studies relevant to the application of CO2 as on the section dealing with plant . should join the opioid family after almost two a human model of anxiety. Psychopharmacol. Bull. centuries. S. Afr. J. Sci. 89, 25–24. 24, 149–153. 24. Alexander F.(1876). In 44. Russel R.W. and Steinberg H. (1955). Effects of 1. Laing M. (2003). Some thoughts on the structure An introduction to Veterinary , 3rd edn, pp. 99–122. Churchill nitrous oxide on reactions to ‘stress’. Quat. J. Exp. and behaviour of N O, laughing gas. S. Afr. J. Sci. 2 Livingstone, Edinburgh. Psychol. 7, 67–73. 99, 109–114. 45. Gillman M.A. and Katzeff I.E. (1988). Does opioid 2. Bowman W.C. and Rand M.J. (1980). Textbook of 25. Reisine T.and Pasternak G. (1996). Opioid analge- uncoupling of adrenal responses mediate opioid Pharmacology, 2nd edn, pp. 39.14–15. Blackwell, sics and antagonists. In Goodman and Gilman’s The anti-stress effects. Med. Sci. Res. 16, 207–209. Oxford. Pharmacological Basis of Therapeutics, 9th edn, ed. 46. Gillman M.A. and Katzeff I.E. (1989). Antistress 3. Dennis D.T. and Turpin D.H. (Eds)(1990). Plant J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. hormonal responses of analgesic nitrous oxide. Physiology, and . Ruddon and A.G. Gilman, pp. 521–555. McGraw- Int. J. Neurosci. 49, 199–202. Maskew Miller Longman, Singapore. Hill, New York. 47. Clark M. and Brunick A. (2003). Handbook of Ni- 4. Hyun J., Chaudhuri G. and Fakuto J.M. (1999). 26. Alexander F.(1976) In An Introduction to Veterinary trous Oxide and Oxygen Sedation, pp. 28–32. Mosby, The reductive metabolism of in Pharmacology, 3rd edn, pp. 184–190. Churchill St Louis. hepatocytes: possible interaction with thiols. Livingstone, Edinburgh. . Metab. Dispos. 27, 1005–1009. 27. Hobbs W.R., Rall T.W. and Verdoorn T.A. (1996). 48. Silver G.A. (1953). Carbon dioxide therapy. Sthn 5. Lipschultz F., Zafiriou O.C, Wofsy S.C., McElroy and . In Goodman and Gilman’s Med. J. 46, 283–286. M.B., Valois F.W. and Watson S.W. (1981). Produc- The Pharmacological Basis of Therapeutics, 9th edn, 49. Brill N.Q., Crumpton E., Eiduson S., Grayson ed. J.G. Hardman, L.E. Limbird, P.B. Molinoff, H.M., Hellman L.I. and Richards R.A. (1959). Rela- tion of NO and N2O by soil nitrifying bacteria. Nature 294, 641–643. R.W. Ruddon and A.G. Gilman, pp. 361–396. tive effectiveness of various components of 6. Smart R.S. and Bloom A.J. (2001). Wheat leaves McGraw-Hill, New York. electroconvulsive therapy. Arch Neurol. Psychiat. emit nitrous oxide during nitrate assimilation. 28. Waller D.D. (1985). Molecular identification of the 81, 627–635. Proc. Natl Acad. Sci. USA 98, 7875–7878. mode of interactions of nitrous oxide with the opiate 50. Zádor J. (1928). Der Lachgas (N2O) – Rausch in 7. Einarsdottir O. and Caughey W.S.(1988). Interac- receptor system. M.Sc. thesis, University of the seiner Bedeutung für Psychiatrie und Neuro- Witwatersrand, Johannesburg. logie. Arch. Psychiatrie 84, 1–71. tions of the anesthetic N2O with bovine cytochrome c oxidase. J. Biol. Chem. 263, 9199– 29. Gamble G.D. and Milne R.J. (1990). Hypercapnia 51. Lichtigfeld F.J. and Gillman M.A. (2003). Another 9205. depresses nociception: endogenous opioids marker for depression. Int. J. Neuropsychopharma- 8. Sowa S., Dong A., Roos E.E. and Caughey W.S. implicated. Brain Res. 514, 198–205. col. 6, 91–92. (1987). The anesthetic nitrous oxide affects di- 30. Clark M. and Brunick A. (1999). Handbook of 52. Lichtigfeld F.J. and Gillman M.A. (1982). The oxygen utilization by bovine heart and bean seed Nitrous Oxide and Oxygen Sedation, pp. 151–155. treatment of alcoholic withdrawal states with mitochondrial . Biochem. Biophys. Res. Mosby, St Louis. oxygen and nitrous oxide. S. Afr. Med. J. 61, Commun. 144, 643–648. 31. Ori C., Ford- F. and London E.D. (1989). 349–351. 9. Falkowski P.G. (1997). Evolution of the nitrogen Effects of nitrous oxide and halothane on mu 53. Gillman M.A. and Lichtigfeld F.J.(2004). Enlarged cycle and its influence on the biological sequestra- and kappa opioid receptors in guinea-pig brain. double-blind randomised trial of benzodiaze- 70, 541–544. pines against psychotropic analgesic nitrous tion of CO2 in the ocean. Nature 387, 272–275. 10. Trudell J.R. (1985). Metabolism of nitrous oxide. In 32. Moody E.J., Mattson M., Newman A.H., Rice K.C. oxide for alcohol withdrawal. Addict. Behav. 29, 1183–1187. Nitrous oxide/N2O, ed. E.I. Eger II, pp. 204–210. and Skolnick P. (1989). Stereospecific reversal of , New York. nitrous oxide analgesia by naloxone. Life Sci. 44, 54. Kripke B.J. and Hechtman H.B. (1972). Nitrous 11. Gillman M.A., Kok L. and Lichtigfeld F.J. (1980). 703–709. oxide for pentazocine addiction and intractable Paradoxical effect of naloxone on nitrous oxide 33. Daras C., Cantrill R.C. and Gillman M.A. (1983). pain. Anesth. Analg. 51, 520–526. analgesia in man. Eur. J. Pharmacol. 61, 175–177. 3(H)-Naloxone displacement: evidence for nitrous 55. Gillman M.A. and Lichtigfeld F.J.(1985). Analgesic 12. Gillman M.A. and Lichtigfeld F.J. (1981). A com- oxide as an opioid agonist. Eur. J. Pharmacol. 89, nitrous oxide: adjunct to clonidine for opioid parison of the effect of morphine sulphate and 177–178. withdrawal. Am. J. 142, 784–785. nitrous oxide analgesia on chronic pain states in 34. Eisele J.H. (1985). Cardiovascular effects of nitrous 56. LaVerne A.A. (1973). Carbon dioxide therapy

man. J. Neurol. Sci. 45, 41–45. oxide analgesia. In Nitrous Oxide/N2O, ed. E.I. Eger (CDT) of addictions. Behav. Neuropsychiat. 4, 13–32. 13. Gillman M.A. and Lichtigfeld F.J. (1981). The II, pp. 126–156. Elsevier, New York. 57. Lichtigfeld F.J.and Gillman M.A. (1982). The similarity of the action of nitrous oxide and 35. Ebert T.J. and Kampine JP. (1989). Nitrous oxide treatment of alcoholic withdrawal states with morphine. Pain 10, 110. augments sympathetic outflow: direct evidence oxygen and nitrous oxide. S. Afr. Med. J. 61, 14. Bredt D.S., Hwang P. M. and Snyder S.H. (1990). from human peroneal nerve recordings. Anesth. 349–351. Localization of nitric oxide synthase indicating a Analg. 69, 444–449. 58. Kripke B.J. and Hechtman H.B. (1972). Nitrous neural role for nitric oxide. Nature 347, 768–770. 36. Guo T., Davis M.F., Kingery W.S., Patterson A.J., oxide for pentazocine addiction and intractable 15. Meller S.T. and Gebhart G.F. (1993). Nitric oxide Limbird L.E. and Maze M. (1999). Nitrous oxide pain. Anesth. Analg. 51, 520–526. α (NO) and nociceptive processing in the spinal produces antinociceptive response via 2B and/or 59. Gillman M.A. and Lichtigfeld F.J.(1985). Analgesic α cord. Pain 52, 127–136. 2C adrenoceptor subtypes in mice. Anesthesiology nitrous oxide: adjunct to clonidine for opioid 16. Gillman M.A. and Lichtigfeld F.J. (1994). NO 90, 470–76. withdrawal. Am. J. Psychiatry 142, 784–785.