1. CARBON MONOXIDE A novel neurotransmitter Prabhat kumar Padhi V-2005-30-12 M.V.Sc. Student Dept of Pharmacology & Toxicology College of Veterinary & Animal Sciences CSKHPKV, Palampur

2. Carbon monoxide & neurotransmission ?!$%@? The word carbon monoxide brings to our mind the thought of a heavily polluted world, deaths of unsuspicious people inside closed rooms and all the bad names that it has earned until now.

3.  Even more interesting is the possibility that this highly toxic molecule may act as a transmitter in the nervous system!  This discovery was triggered by the elucidation of the neurotransmitter role of the more toxic Nitrogen monoxide (NO).  Recent advances in the neuroscience field have suggested diverse roles for CO in the nervous system.

4.  In Greek and roman times the role of carbon monoxide as a poisonous gas was Known. It was used for executions.  Claude Bernard first described the mechanism of toxicity of CO in 1857.  In 1927 the effect of CO on tissue uptake of O 2 was demonstrated.  Warburg used yeast tissue cultures to show that cellular uptake of O 2 was decreased by large doses of CO. Brief history of toxic CO Brief history of toxic CO

5. Brief history of neurotransmitter CO  1952 Sj ö strand reported the endogenous synthesis of CO.  1968 heme oxygeanse was identified.  Mid 1980 isoforms of HO were discovered.  1993 role of CO as neurotransmitter by Verma et al.  In 1997 HO3 was discovered.

6. Brief history of neurotransmitter CO  On the right is the photograph of Solomon H. Snyder ( Johns Hopkins University School of Medicine).  He is one among the foremost authorities in this field.

7. Carbon monoxide as a toxic gas

8. Sources of carbon monoxide Exogenous sources Incomplete combustion of organic fuel Cigarette & household smoke Vehicular & industrial pollution

9. Mechanism of toxicity  210 times more affinity for Hb than O 2.  Oxyhemoglobin dissociation curve shifted to left.  Decreases tissue uptake of O 2.  Decreases function of cytochrome P 450

10. Signs of toxicity  Colorless, odourless gas not detected by human senses.  Low conc- cardiovascular and neurobehavioural effects  High conc- coma and death  Treatment hyperbaric O 2. (Haldane 1895)

11. Carbon monoxide the neurotransmitter

12. Sources of carbon monoxide Endogenous sources Product of heme catabolism Lipid peroxidation Photo-oxidation, xenobiotics

13. Synthesis of CO  For the endogenous synthesis of CO heme is the substrate.  Also required for this process are  NADPH  cytochrome P 450  enzyme heme oxygenase (HO).  Endogenous heme is localized in neurons. In fact heme is synthesized in every nucleated cell of the body.

14. Synthesis of CO There are 3 isoforms of HO  HO1 (inducible), found in many tissues like lungs, spleen ageing RBC etc.  HO2 (constitutive) found in brain, neurons in GIT, blood, testes.  HO3 (constitutive) found in rats. It does not have potent enzymatic activity

15. Synthesis of carbon monoxide Heme Biliverdin Bilirubin NADPH, H + Biliverdin reductase Fe 2+ CONADP3H2O NADPH,3O2, HO1/2 p450 reductase

16. Regulation of HO1 Transcriptionally upregulated by  Synergistic induction. Induction and function of HO1 parallels with physiological activity of a given stimulus. Ex: IL-1, Caffeic acid, Curcumine  Antagonistic induction of HO1 is to counteract the action of the stimuli in a feed back mechanism. Ex: Serum, Angiotensin

17. Regulation of HO2 A. mGluR1 B. Phosphorylation C. Calcium-calmodulin

18. Fate of endogenous Carbon monoxide in the body Endogenously produced CO is removed by the following means Endogenously produced CO is removed by the following means  Expiration  Scavenging  Oxidation

19. Findings that support role of CO as a neurotransmitter Work on Olfactory receptor neurons Work on Olfactory receptor neurons  HO inhibitors reduce basal cGMP levels & block generation of cGMP in response to odorants in the olfactory receptor neurons.  This presents the strongest evidence favoring the role of CO in the nervous system.

20. Hyperpolarizing factor in the GIT  HO2 null mice have depolarized smooth muscle cells and the membrane potential in the gut is abolished.  Regions of the canine GIT that are more hyperpolarized generate more CO and exhibit more HO2 activity.  Genomic deletion of biosynthetic enzymes, Antiserum against HO, inhibition of enzyme HO have also demonstrated the possible roles of CO as a neurotransmitter. Other findings

21. HO2 is extensively localised in HO2 is extensively localised in  Olfactory bulb, Hippocampus area  Gastrointestinal tract.  Genitourinary tract  Blood vessels, Glomus cells of carotid body  Trachea Localization of HO2

22. Differences from classical neurotransmitters Classical neurotransmitters Gaseous neurotransmitters Are synthesized and stored in vesicles Are synthesized fresh and not stored Act via receptors like muscarinic, nicotinic etc. Too small to have receptors. Directly diffuse across cell membrane and act on molecules. Exp: Ach, epinephrine etcExp: CO, NO, H 2 S

23. Targets of CO  Soluble Guanylyl cyclase (sGC)  Ion channels  K + Channels (K Ca and K ATP )  Na + channels  Ca 2+ channels  Hemoprotein targets and others  P 38 MAPK

24. Targets of CO

25. Physiological roles In the circulatory system  Carbon monoxide mediates endothelial derived vasorelaxation.  It acts in concert with NO in inducing vasorelaxation.

26. Physiological role Urogenital system  HO2 occurs throughout the autonomic nervous system. HO2 neuronal staining occurs in the pelvic ganglion and nerve fibers that innervate the bulbospongiosus and related muscles.  CO also plays a role in micturition. It is localised in detrussor muscle of bladder, urethral sphincter.

27. Physiological role Gastrointestinal tract  HO2 activity is prominent in internal anal sphincter area.  Also prominent in the myenteric plexus.  Also in the Interstitial cells of Cajal (ICC), the pace maker of the small intestine.  Also found in ganglionic cells located near islets of Langerhans and in liver.

28.  CO plays a role in NANC transmission in the GIT.  Role in pacemaker activity,choleresis, insulin release.  It is an endogenous hyperpolarizing factor in the GIT.  It mediates the VIP associated NANC neurotransmission

29. Physiological role In the nervous system CO plays roles in the following functions  Odor response  Vision  Hearing  Thermal regulation  Nociception  Chemoreception  LTP, memory

30. Physiological role Hormones  Mediates release of GnRH, Neuropeptide Y from hypothalamus.  Release of Insulin from islets of Langerhans. Neuromuscular junction  The presence of HO2 immunoreactivity in the rat neuromuscular junction suggests that it may act as a post and pre-synaptic neurotransmitter in the NMJ.

31. Physiological role Circadian rhythm  CO acts on NPAS2 a mammalian transcription factor that regulates circadian rhythm.  It has also been linked to the suprachiasmatic nucleus in the brain. CO has got antiinflamatory (acts on COX) and anti-apoptotic activities. These activities can be exploited in preventing graft rejection.

32. Cotransmission with NO CO and NO act together additively in the  Gastrointestinal system  Nervous system  Genitourinary system Many of the functions of NO are dependent upon the presence of CO.

33. Target of NO & CO

34. Comparison between NO & CO nNOSHO2 BiosynthesisCatalyzes a mixed oxidation of arginine to form the diatomic gas radical, NO. Catalyzes a mixed oxidation of heme to form the inert diatomic gas, CO. Gene isoforms iNOS, inducible nNOS, eNOS, constitutive HO1, inducible HO2, constitutive Subcellular localization Dendrites, axons, endoplasmic reticulum, and cytosol Exclusively on the endoplasmic reticulum

35. Activation nNOSHO2 Calcium/calmodulinProtein kinase C Soluble guanyl cyclase Role in blood vessels Can directly alter protein function by S- nitrosylation. Other targets? Originally discovered as endothelial- derived relaxation factor Like eNOS and nNOS, HO2 is present in both the endothelium and the surrounding adventitial neurons. Role in gastrointestin al tract Nonadrenergic, noncholinergic neurotransmitter, most prominently in the pylorus Nonadrenergic, noncholinergic neurotransmitter, most prominently in the internal anal sphincter

36. nNOSHO2 Role in urogenital tract nNOS + neurons innervate the corpus cavernosum. NOS inhibitors prevent penile erections. HO2 + neurons innervate the bulbospongiosus muscles. Ejaculation is reduced in HO2/ mice. BehaviornNOS/mice are aggressive and inappropriately mount females, regardless of estrus stage. ? NeurotoxicityActivation of nNOS augments toxicity by generation of a free radical. Activation of HO2 protects against toxicity by quenching free radicals.

37. Pathophysiology Neurodegeneration and brain disorders  HO/CO system has been implicated in various neurodegenerative diseases like  Alzheimer's disease  Parkinson's disease  Amyotrophic lateral sclerosis  This system has also been found altered in seizure disorders.

38. Pathophysiology CO in cardiovascular pathophysiology  Hypertension In hypertension CO/HO system plays a role in following conditions  Hypertension induced by HO inhibitors  Spontaneously hypertensive rats  Salt induced hypertension  Angiotensin-II induced hypertension  Renovascular hypertension  Portal hypertension  Cardiac hypertrophy and heart failure

39. Pathophysiology In addition role of CO/HO system can be traced to disorders like  Neurotoxicity  Genitourinary diseases  GIT disorders

40. Pathophysiology  Carbon monoxide in transplantation  Survival of Allograft  Survival of Xenograft  Carbon monoxide in apoptosis and cell proliferation  Vascular smooth muscles  Endothelial cells  Other types of cell

41. Carbon monoxide in lungs disease

42. Therapeutic applications of carbon monoxide Carbon monoxide can be applied for therapeutic purposes by  Up regulation of enzyme HO  Down regulation of enzyme HO  Inhalation of CO  Use of CO releasing compounds  Use of prodrugs to release CO

43. Roles for future  Prevention of graft rejection  Fight against inflammation  Correction of hypertension and lung diseases.  Treatment of diseases of urogenital system.  Treatment of GIT diseases (Irritable bowel syndrome)

44. Thank you