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![Arginine Paradox
Intracellular [Arginine] = 0.8-2.0 mM
Plasma [Arginine] = 100-150 µM
Km of Arginine for cNOS = 2.9 µM
Exogenous Arginine is beneficial in endothelial-dependent vasodilation.
• Intravenous infusion of L-arginine in healthy humans significantly increased
vasodilation (Bode-Boger et al., 1998)
• Oral L-arginine improves endothelial function in healthy adults older than
70 years (Bode-Boger et al., 2003)
• Oral L-arginine improves endothelial dysfunction in patients with essential
hypertension (Lekakis et al., 2002)
• In cystic fibrosis patients, oral L-arginine was shown to enhance lower
airway NO formation (Grasemann et al., 2005)](https://image.slidesharecdn.com/03a13fa6-b0f1-4f08-b6e0-7071fb43c937-160317233444/85/Agmatine-7-320.jpg)
![In vivo studies in humans
(Bode-Boger et al., 1998)
L-arginine [Plasma arg], mM Peripheral resistance
6 g 0.82 ± 0.059 No effect
30 g 6.22 ± 0.4 Significant decrease](https://image.slidesharecdn.com/03a13fa6-b0f1-4f08-b6e0-7071fb43c937-160317233444/85/Agmatine-17-320.jpg)
![L-arginine triggered [Ca2+
]i release
0 400 800 1200 1600
0.75
0.80
0.85
0.90
0.95
1.00
1.05
0 300 600 900 1200 1500
0.75
0.80
0.85
0.90
0.95
1.00
1.05
Fluor.ratio(340nm/380nm)
5 mM L-arginine
Time, sec Time, sec
2 nM Rauwolscine
L-arg + Rauwolscine](https://image.slidesharecdn.com/03a13fa6-b0f1-4f08-b6e0-7071fb43c937-160317233444/85/Agmatine-20-320.jpg)
![Conclusions
• The NO formation (from both L-arginine and D-arginine) was inhibited by I-
receptor and α-2 AR antagonists, indicating the possible involvement of
receptor(s) in the NO formation.
• The involvement of G-proteins was indicated by the inhibition of NO by
pertussis toxin, a specific inhibitor of Gi/o-proteins.
• The addition of L-arginine yielded a transient increase in [Ca2+
]i within 1 min
and Ca2+
dependence of NO formation was demonstrated using inhibitors of
intracellular Ca2+
signaling.
• The agmatine directly activated endothelial NO at low concentrations as
compared to arginine and this could be attenuated by α-2 AR antagonist,
rauwolscine.
• L-arginine-initiated dilation of isolated gracilis vessels is being inhibited by α-
2 AR antagonist, rauwolscine and thus establishing the physiological
relevance of our central hypothesis.](https://image.slidesharecdn.com/03a13fa6-b0f1-4f08-b6e0-7071fb43c937-160317233444/85/Agmatine-37-320.jpg)
![ARGININE ,HISTIDINE AND BRANCHED CHAIN AMINO ACIDS [Autosaved] HISTIDINE AND ...](https://cdn.slidesharecdn.com/ss_thumbnails/argininehistidineandbranchedchainaminoacidsautosavedhistidineandproline-250321163551-9c372010-thumbnail.jpg?width=640&height=640&fit=bounds)
Agmatine
- 1. Receptor-Mediated Activation ofNitric Oxide Synthesis by Arginine in the vasculature MAHESH JOSHI
- 2. Nitric Oxide: Amarvelous molecule
- 3. Historical Background • 1977Nitric Oxide activates sGuanylate Cyclase – Murad, F. • 1980 Endothelium derived relaxing factor (EDRF) discovered – Furchgott, RF. • 1987 EDRF is Nitric Oxide – Ignarro, LJ., and Moncada, S. • 1992 Molecule of the Year – Science Journal • 1998 Nobel Prize (Physiology and Medicine) - Murad, Furchgott and Ignarro
- 4. Blood vessel anatomy Smoothmuscle cells Endothelial cells NO NO
- 5. Summary of NitricOxide ChemistrySummary of Nitric Oxide Chemistry Prooxidant and Antioxidant ReactionsProoxidant and Antioxidant Reactions .. NONO NONO22 .. ONOOONOO–– RNORNO NONO–– Me-NOMe-NO NONO++ antioxidantantioxidant prooxidantprooxidant one-electronone-electron redoxredox one-electronone-electron redoxredox O2 O2 .– R.Me + e– – e–
- 6. Stereospecificity of substratefor eNOS D-arginine is not a substrate Arginine Transport • Mediated by specialized carrier systems: y+ , b+ , B+ and y+ L • Cationic amino acids share the same carrier systems • In endothelial cells, three genes Cat1, Cat2, and Cat3 encode system y+ COO- C (CH2) 3 NH C H2N H NH2+ +H3N L-Arginine NOS COO- C (CH2) 3 NH C H+H3N N + H2N H OH Nω -Hydroxy-L-arginine COO- C (CH2) 3 NH H+H3N + NO . NOS C O NH2 L-Citrulline
- 7. Arginine Paradox Intracellular [Arginine]= 0.8-2.0 mM Plasma [Arginine] = 100-150 µM Km of Arginine for cNOS = 2.9 µM Exogenous Arginine is beneficial in endothelial-dependent vasodilation. • Intravenous infusion of L-arginine in healthy humans significantly increased vasodilation (Bode-Boger et al., 1998) • Oral L-arginine improves endothelial function in healthy adults older than 70 years (Bode-Boger et al., 2003) • Oral L-arginine improves endothelial dysfunction in patients with essential hypertension (Lekakis et al., 2002) • In cystic fibrosis patients, oral L-arginine was shown to enhance lower airway NO formation (Grasemann et al., 2005)
- 8. Importance of ExtracellularArginine: Plasma Levels in Disease Conditions • Low plasma arginine levels in children with cerebral malaria (Lopansri et al., 2003) • In newborns with persistent pulmonary hypertension, plasma arginine utilization for whole body NO synthesis was reduced (Castillo et al., 1995) • Women with high resistance placental circulation at risk of pre- eclampsia have raised plasma ADMA levels (Savvidou et al., 2003) • Decreased arginine bioavailability and increased arginase activity in asthma (Morris et al., 2004)
- 9. Explanations for arginineparadox • Endogenous eNOS inhibitors example: asymmetric dimethylarginine (ADMA) • Compartmentalization of arginine eNOS coupled to caveolae • Antioxidant effects of arginine L-arginine attenuates O2 - formation • Structural similarity to receptor ligands Imidazoline/alpha-2 receptor ligands have guanidinium moiety
- 10. Structural similarity: L-arginineand imidazoline/α-2 receptor ligands Generated by CamScanner
- 11.
- 12. Supporting Evidence forReceptor Hypothesis
- 13. Both arginine andα-2 adrenoceptor agonists cause endothelium-dependent relaxations UK-14304 induced dilation of aortic rings from SHR and normotensive rats (Sunano et al. 1996) Clonidine dose-dependently elicited rat mesenteric artery dilation (Figueroa et al. 2001) Injection of Moxonidine reduced mean arterial pressure in Wistar rats (Moreira et al. 2004)
- 14. Imidazoline and α-2adrenoceptors • There is very little knowledge about the pharmacological functions of I- receptors. • But they are believed to be involved in the control of blood pressure, eating disorders in rats, mental pathological conditions and neurodenerative disorders. • α-2 adrenoceptors have been widely studied due to their role in control of blood pressure and blood flow, neural modulation, digestion, reproduction, endocrine and metabolic processes. • Endothelial cells do contain α-2 adrenoceptors. However, their abundance in the cell membranes is not known.
- 15. Experimental design: Effectof receptor antagonists Cell cultures (HUVEC) L-arg ± antagonists 15-30 min, 37° C Supernatants Griess reaction NO2 - , NO3 -
- 16. Inhibition with imidazoline/α-2AR receptor antagonist None Arginine,5mM +L-NAME,5mM 0.0 0.4 0.8 1.2 1.6 NO 2 - +NO 3 - ,µM * *A NO 2 - +NO 3 - ,µM None 5mML-Arginine +10µMIdazoxan 0.0 0.2 0.4 0.6 0.8 1.0 # #B
- 17. In vivo studiesin humans (Bode-Boger et al., 1998) L-arginine [Plasma arg], mM Peripheral resistance 6 g 0.82 ± 0.059 No effect 30 g 6.22 ± 0.4 Significant decrease
- 18. Inhibition with α-2AR antagonist None 5mML-arginine +0.2nMRauwolscine 0.0 0.2 0.4 0.6 0.8 1.0 1.2 NO 2 - +NO 3 - ,µM * * A
- 19. Receptor antagonists inhibitedD-arg mediated NO synthesis None 5mMD-Arginine +0.2nMRauwolscine +50µMIdazoxan 0.0 0.2 0.4 0.6 $ NO2 - +NO3 - ,µM * #
- 20. L-arginine triggered [Ca2+ ]irelease 0 400 800 1200 1600 0.75 0.80 0.85 0.90 0.95 1.00 1.05 0 300 600 900 1200 1500 0.75 0.80 0.85 0.90 0.95 1.00 1.05 Fluor.ratio(340nm/380nm) 5 mM L-arginine Time, sec Time, sec 2 nM Rauwolscine L-arg + Rauwolscine
- 21. Role of Ca2+ inL-arginine actions None 5mML-arginine +5mMLanthanum 0 200 400 600 800 1000 B. Radiolabeled 45 Ca 2+ 45Ca2+,CPM # # None 5mML-arginine +10mMEGTA 0.0 0.1 0.2 0.3 0.4 0.5 NO2 - +NO3 - ,µM * *
- 22. Inhibitors of Ca2+ signalingattenuate NO synthesis None 5mML-arginine +5µMNifedipine +3µMU-73122 5µMNifedipine 3µMU-73122 0.0 0.5 1.0 1.5 2.0 2.5 3.0 NO3 - +NO2 - ,µM
- 23. α-2 AR agonistactivates NO synthesis 0.001 0.05 0.5 0.00 0.25 0.50 0.75 1.00 NO 3 - +NO 2 - ,µM Dexmedetomidine, nM * *
- 24. Does G-protein mediateL-arginine actions?
- 25. CYTOPLASM Extracellular Space GPCR Endoplasmic Reticulum inactiveGq GDP active Gq GTPGDP inactive PLC active PLC DAG IP3 Ca2+ Ca2+ IP3 GTP Arginine eNOS NO Ca2+ Ca2+
- 26. Pertussis toxin inhibitsNO formation None 5mML-arginine +200ng/mlPTx +400ng/mlPTx 0.0 0.4 0.8 1.2 1.6 2.0 # # * * NO 2 - +NO 3 - ,µM
- 27. Rat gracilis vesseldilation L-arginine,1mM +0.2nMRauwolscine -4 0 4 8 12 16 Max.dilation,µm *
- 28. Arginine as agonist:A problem Arginine Agmatine Ca2+ eNOS NO Arginine Decarboxylase (ADC)Receptor Extracellular Space Cytoplasm 1. Millimolar quantities of Arginine are required for activation of NO 2. Both L-and D-arginine activated NO synthesis Alternative hypothesis: Agmatine as agonist
- 29. Is there amammalian ADC? • Its activity was detected in rat brain (Don Reis group, Science 1994) • Partial cloning of ADC from rat kidney was achieved (Morrissey et al. 1995) • ADC activity was also demonstrated in membrane enriched fractions of brain, liver and kidney cortex and medulla (Lortie et al. 1996) • Using 15 N-labeled arginine and GC-MS, ADC reaction was documented in isolated mitochondria from perfused rat liver (Horyn et al., 2005)
- 30. Agmatine: Relevance toNO system • Agmatine is bioactive in a number of tissues and is present in serum • A structural analog of L-arginine but not a substrate for Nitric Oxide Synthases • Binds to α-2 adrenoceptor and imidazoline receptors in the brain • It acts as a systemic vasodilator in rat, when injected i.v and in isolated aorta
- 31. Agmatine activates NOsynthesis None 1µMAgmatine 10µMAgmatine +0.2nMRauwolscine 0.0 0.2 0.4 0.6 0.8 NO 2 - +NO 3 - ,µM # #
- 32. Untreated 10 µMAgmatine 10 µM Agm + 1 mM L-NAME Agmatine activation of cellular NO : DAF-FM diacetate assay 4-amino-5-methylamino-2’,7’-difluoro fluorescein diacetate; DAF-FM DA
- 33. Q: If argininedecaboxylates via ADC to agmatine, why does it take only micromolar agmatine whereas millimolar quantities of arginine are required to activate NO synthesis? A: Back to Basics Km of Arginine for ADC: 0.75 - 46 mM (Horyn et al., 2005; Regunathan,S and Reis, DJ., 2000)
- 34. Rat Mesenteric ArteryRelaxation DFMA, arginine decarboxylase inhibitor EC50 Arginine: 5.8±0.7 mM ; Agmatine; 139±12 µM
- 35. Mediation of alpha-2AR and G-protein RX821002, alpha-2 antagonist PTx, G-protein inhibitor
- 36. High Salt vsNormal Salt: NO and Gene Expression NO levels RT-PCR
- 37. Conclusions • The NOformation (from both L-arginine and D-arginine) was inhibited by I- receptor and α-2 AR antagonists, indicating the possible involvement of receptor(s) in the NO formation. • The involvement of G-proteins was indicated by the inhibition of NO by pertussis toxin, a specific inhibitor of Gi/o-proteins. • The addition of L-arginine yielded a transient increase in [Ca2+ ]i within 1 min and Ca2+ dependence of NO formation was demonstrated using inhibitors of intracellular Ca2+ signaling. • The agmatine directly activated endothelial NO at low concentrations as compared to arginine and this could be attenuated by α-2 AR antagonist, rauwolscine. • L-arginine-initiated dilation of isolated gracilis vessels is being inhibited by α- 2 AR antagonist, rauwolscine and thus establishing the physiological relevance of our central hypothesis.
- 38. Future direction: location,location, location Caveola Gα-2 AR Agmatine IP3 Ca2+ eNOS L-Arg NO Membrane Ca2+