Both the smooth muscle and the endothelium of blood vessels have cholinergic innervation
Cholinergic nerve endings Adrenergic nerve endings
Smooth muscle endothelium
Preparation of rabbit aortic wall strips
The endothelium is required for the relaxing effect of acetylcholine on vascular smooth muscle
Smooth muscle tension
Norepinephrine
Acetylcholine
Wash (removal of norepinephrine)
Adapted from Furchgott et al, Nature 288:373 (1980)
The relaxing effect of the endothelium is mediated by a diffusible substance
Smooth muscle tension
Norepinephrine
Acetylcholine
Wash
Adapted from Furchgott et al, Nature 288:373 (1980)
Identification of ‘endothelial relaxing factor’ (EDRF) as NO (1): Both NO and EDRF react with hemoglobin
O2
N N
NO
N N
Fe2+ N N NO3NO
N N
Fe3+ N N
NO
N N
Fe2+
N N
Fe2+ N N
Identification of ‘endothelial relaxing factor’ as NO (2): The effects of both NO and EDRF are potentiated by superoxide dismutase
Superoxide dismutase H+
O2 + e•O-O-
½ O2 + ½ H2O2
•N=O
-O-O-N=O
(peroxynitrite)
Identification of ‘endothelial relaxing factor’ as NO (2): Direct measurement of endogenous NO by chemiluminescence
•NO
+ O3
•NO 2
+ O2
h•n
N2O4
How NO is generated
H2 N
NH2 HN
+
H2 N
N HN
OH
H2 N
O HN
+ NO
H2 N
C H
COOH
H2 N
C H
COOH
H2 N
C H
COOH
NADPH + O2 NADP+ +H2O
½ (NADPH + H+ + O2) ½ NADP+
NH2-
H4B
Heme
CaM
FMN
FAD
NADPH
-COOH
NADPH
e-
FAD
Heme H4B
FMN CaM Arg H4B Arg Heme
CaM FMN FAD
e-
NADPH
NOS occurs in different forms and tissues
Ca++
Transcriptional Induction
eNOS (endothelial cells)
+
-
nNOS (nerve cells)
+
-
iNOS (macrophages)
-
+
NO reaches its signaling target by diffusion of across multiple cell membranes
Cholinergic nerve terminals Ca++ NO
Ca++ NO
Endothelial cells
Smooth muscle cells
Intracellular biochemical effects of NO (1)
NO Receptor guanylate cyclases
GTP
sGC cGMP
PDE
cAMP
PKG cGMP-gated channel
AMP
Proteins Proteins-P
Guanylate cyclase: Allosteric activation by NO binding to heme
N 2+ N N Fe N
N
His
N H
NO
N 2+ N N Fe N
GTP
N
cGMP
His
N H
Protein kinase G effects on the vascular smooth muscle (1): Phosphorylation of myosin light chain phosphatase
PKG ATP MLC-P’ase
ADP MLC-P’ase-P P
P
ADP
MLCK
ATP
Protein kinase G effects on the vascular smooth muscle (2): Phosphorylation of the IP3 receptor Ca++ channel
P ATP
ADP MLCK
PKG
ATP
Ca++
IP3-R
P
ADP
CaM
Ca++
ER
Intracellular biochemical effects of NO (2): S-nitrosylation and transnitrosylation
R-SH + N=O R-S-N-O-H + O2 ( O-O- + N=O R-S-N-O-H R-S-N=O + O-O- + H+ O=N-O-O- )
R1-S-N=O + R2-SH
R1-SH + R2-S-N=O
Immunohistochemical detection of protein S-nitrosylation
Uncoloured, soluble substrate
Enzyme
Coloured, insoluble product Protein-S-NO
NOS-dependent S-nitrosylation of proteins in vascular endothelium and smooth muscle
From J. Biol. Chem. 277:9637-9640 (2002)
Identification of S-nitrosylated proteins (1)
-SH -S-NO -S -S -S-S-CH3 -S-S-biotin -S -S Enzyme Streptavidin
MMTS
-S-S-CH3 -S-NO -S -S NO
Biotin-HPDP -S-S-CH3 coloured product
Ascorbate
-SH -S -S
S-Nitrosylation of neuronal cell lysates in vitro
Protein-SH + Glutathione-S-NO Protein-S-NO + Glutathione-SH
E
Data from Nat Cell Biol. 3:193-7 (2001)
Identification of S-nitrosylated proteins ex vivo (1)
-S-CH3
-S-S-biotin -S -S
b-ME
CH3-SH
HS-biotin
-SH -SH -S -S
Starting material
Column-eluted material
Identification of S-nitrosylated proteins ex vivo (2)
From Nat Cell Biol. 3:193-7 (2001)
More NO-releasing drugs
NO+
ONO2
H3C HC H3C C H2 C H2 O N O
O
O
CN-
CN-
Fe2+
ONO2
CN-
CNCN-
Amylnitrite
Isosorbide dinitrate
Nitroprusside
Summary of established and potential NO signaling pathways
From Cell. Mol. Life Sci. 55 (1999) 1036-1042
NO is a ‘killing effector’ in macrophages
Macrophage
Microbe
Arg NOS NO O2O=N-O-O-
O2-, H2O2
The first NO-releasing drug
H2C HC H2C
O O O
NO2 NO2 NO2
Mechanism of NO release from drug molecules
• • Nitrates react with sulfhydryl (-SH) groups, but the uncatalyzed reaction is too slow to account for the rapid (almost immediate) onset of clinical effect Enzymatic activities that facilitate release have been described and preliminarily characterized: • Membrane-associated • Inactivated by covalent SH modification – suggests active-site cysteine • Identity, physiological function, and significance in vivo unknown Nitroprusside reacts faster with sulfhydryls, may not need catalysis for NO release
•
NOS inhibitors (1)
CH3
H2 N
NH2 HN
+
NO2 HN
NH2
+
HN
NH2 HN
+
HN
H2 N
C H
COOH
H2 N
C H
COOH
H2 N
C H O
OCH3
arginine
N-methylarginine
N-nitroarginine methylester
NOS inhibitors (2)
CH3 S
NH2
+
S C H2 N
C2 H5 NH
HN
H2 N
C H
COOH
S-ethyl-thioisourea
S-methyl-isothiocitrulline
NOS inhibitors (3)
S NH N H N H
NH NH
Cl
ARL 17477 (nNOS-selective)
6-cyclohexyl-2-iminopiperidine (iNOS-selective)
cGMP action is terminated by a cGMPselective phosphodiesterase
NO
GTP
sGC
cGMP
PDE 5
effector proteins
GMP
cGMP action is terminated by a cGMPselective phosphodiesterase
NO GTP
CH3 H2C O HN
O
CH3 N N
sGC cGMP
effector proteins
N H2C CH2 O S N O N CH3 CH3
PDE 5
GMP
Sildenafil (Viagra)