3’,5’-cAMP CELL 3’,5’-cAMP 5’-AMP ADO ATP AC Ecto -3’,5’-PDE Ecto-5’-NT Paracrine Autocrine Tr ansporter Extracellular 3’,5’-cAMP- Adenosine Pathway Jackson, E.K.: Annual Review of Pharmacology and Toxicology 31: 1-35, 1991.
Jan 21, 2016
3’,5’-cAMP
CELL
3’,5’-c
AMP
5’-AMP
ADO
ATP
AC
Ecto-3’,5’-PDE
Ecto-5’-NT
ParacrineAutocrine
Transp
orter
Extracellular 3’,5’-cAMP-AdenosinePathway
Jackson, E.K.: Annual Review of Pharmacology and Toxicology 31: 1-35, 1991.
UPLC
ION SOURCE
1st Quadrupole
PARTICLE DETECTOR
Separates Analytes in Time Domain According To Their Physicochemical Properties
Electrospray Ion Source to Gently Ionize Analytes In Sample to Generate Parent Ions
Serves as a Mass Filter to Pass Only the Parent Ion of Interest to Next Part of Mass Spec
2nd QuadrupoleServes as a Collision Cell to Fragment Parent Ion
To Daughter Ions
3rd QuadrupoleServes as a Mass Filter to Pass Only the Daughter
Ion of Interest to Particle Detector
Advantage: Very specific, accurate and sensitive assay
Ultra-Performance Liquid ChromatographCoupled to a Triple Stage Quadrupole Mass Spectrometer
Isolated Perfused Rat Kidney
Tyrode’sSolution
PumpDrugsBubble
Trap
Physiograph
Ureter
Artery
Vein
H2O37°C
WaterCirculator
KIDNEY
Pressure Transducer
Applied LC-MS/MS assay to measure 3’,5’-cAMP in renal venous perfusatefrom isolated, perfused rat kidneys
0 1 2 3 4 5 6 7 8
0
10
20
30
40
50
60
70
80
90
100
3’,5’-cAMP
What the #!%&
is this?!
Rela
tive
Abun
danc
e(F
ull S
cale
: 6.5
x 1
03 )
Retention Time (minutes)
330 m/z (3’,5’-cAMP + H+) → 136 m/z (Adenine + H+)
The unknown and 3’,5’-cAMP had same m/z
Fragmentation of the unknown and 3’,5’-cAMP yielded daughter ions with
same m/z
N
HNN
NH+
NH2
OP
O
O
HO
O
HO
N
NN
NH+
NH2
H
H
OHO
O
HO
N
NN
N
NH2
O
P
O
3’,5’-cAMP
O
N
NN
N
NH2
HO
O O
P
O
HO
HYPOTHESISUnknown = 2’,3’-cAMP
0 1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
Rela
tive
Abun
danc
e(F
ull S
cale
: 1.3
x 1
04 )
Retention Time (minutes)
330 m/z → 136 m/z3’,5’-cAMP
2’,3’-cAMP
0 1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
Unknown
330 m/z → 136 m/z
Rela
tive
Abun
danc
e(F
ull S
cale
: 1.0
x 1
04 )
Renal Venous Perfusate From Isolated, Perfused Rat Kidney: During Isoproterenol
Injected authentic 2’,3’-cAMP into LC-MS/MSand monitored the 330 m/z to 136 m/z transition
Ren, J., Mi, Z., Stewart, N., and Jackson, E.K.: Journal of Pharmacology and Experimental Therapeutics 328: 855-865, 2009.
CELL2’,3
’-cAMP
2’-AMP + 3’-AMP
ADO
mRNA
Ecto-2’,3’-PDEs
Ecto-2’/3’-NTs
ParacrineAutocrine
2’,3’-cAMP
Transp
orter
RNases
Extracellular 2’,3’-cAMP-AdenosinePathway
Jackson, E.K., Ren, J., and Mi, Z. Journal of Biological Chemistry 284, 33097-33106, 2009
2’,3’-cAMP, 3’-AMP and 2’-AMP Are Biological Active
O
N
NN
N
NH2
HO
O O
P
O
HO
2’,3’-cAMP 2’,3’-cGMP
HN
NN
N
OH
N
HN
NH2N
HN
O
2’,3’-cIMP
NH
O
HN
O
2’,3’-cUMP
NH
O
N
H2N
2’,3’-cCMP
All possible 2’,3’-cNMPs, except 2’,3’-cTMP, exist
GuanineUracil
HypoxanthineCytosin
e
Bacteria --- Plants --- Animals --- Humans
Confirms mRNA as source
Conserved, therefore important !
CSF Levels of 2’,3’-cAMP TBI Patients
Renal Venous Perfusate Levels of 2’,3’-cAMP in
Energy-Deprived Kidneys
Even Tobacco Leaves Release 2’,3’-cAMP in Response to Injury!!
Big Questions is: Does the Release of 2’,3’-cAMP Promote or Ameliorate Organ Injury?
CELL2’,3
’-cAMP
2’-AMP + 3’-AMP
ADO
mRNA
Ecto-2’,3’-PDEs
Ecto-2’/3’-NTs
TissueProtection
2’,3’-cAMP
Transp
orter
RNases
mPTPs
Apotosis
Azarashvili et al.AJP-Cell 2009, 296:1428
Hypothesis: 2’,3’-cAMP Release Ameliorates Organ Injury
How to test this hypothesis?
Need somewayto block the
pathway!
2’,3’-Cyclic Nucleotide-3’-Phosphodiesterase (CNPase)
Myllykoski et al.JMB 2013, 425: 4307
Crystallographic Analysis of the Reaction Cycle of 2 ,3 -Cyclic ′ ′Nucleotide 3 -Phosphodiesterase, a Unique Member′
of the 2H Phosphoesterase Family
Adenosine
mRNA
2’,3’-cAMP
2’,3’-cAMP
2’-AMP
Opening of mPTP
(Pro-Apoptotic;
Pro-Necrotic)
Adenosine 2’-AMP
(Cell Membrane)
CNPase
CNPase
Does Attenuating the 2’,3’-cAMP-Adenosine PathwayEnhance or Promote Organ Injury?
CNPase = 2’,3’-Cyclic Nucleotide-3’-Phosphodiesterase
XX
CNPase -/- Mouse Expresses Neurodegeneration With Age
WHAT ABOUT THE 2’,3’-cAMP-ADENOSINEPATHWAY BRAIN INJURY?
CNPase 1CNPase 2
GAPDH
CNPase +/+ at 24 hours post injury CNPase -/- at 24 hours post injury
CNPase +/+ at 7 days post injury CNPase -/- at 7 days post injury
CNPase 1 (46 kDa)
PGVSMCsGMCs
Whole Kidney
MWMarkers
20 kDa
30 kDa
40 kDa
50 kDa60 kDa
80 kDa100 kDa120 kDa
CNPase 2 (48 kDa)
β-actin (42 kDa)
22.76 ± 0.10 24.25 ± 0.11 Ct for CNPaseGMCs PGVSMCs
14.58 ± 0.04 15.11 ± 0.11 Ct for β-actin
IS CNPase Expressed In the Kidney?
Examined CNPase Expression in Rat Whole Kidney, GMCs and PGVSMCs
MWMarkersPGVECsCDCsPTCsTALCs
MWMarkers
CNPase 1 (46 kDa)CNPase 2 (48 kDa)
β-actin (42 kDa)
20 kDa
30 kDa
40 kDa50 kDa60 kDa
80 kDa100 kDa120 kDa
23.64 ± 0.24 23.30 ± 0.06
TALCs PTCs
23.34 ± 0.01 24.32 ± 0.01
CDCs PGVECs
Ct for CNPase
Ct for β-actin 14.24 ± 0.20 14.88 ± 0.02 15.15 ± 0.04 16.28 ± 0.01
IS CNPase Expressed In the Kidney?
Examined CNPase Expression in RatTALCs, PTCs, CDCs and PGVECs
IS CNPase Expressed In the Kidney?
Darlot et al., Topology of Schwann cells and sympathetic innervation along preglomerular vessels: a confocal microscopic study in protein S100B/EGFP transgenic mice.
Am J Physiol Renal 295: F1142-1148, 2008.
Is CNPase Involved in the Metabolism of 2’,3’-cAMP in the Intact Kidney?
Isolated and perfused kidneys from CNPase +/+ vs -/- mice, administered 2’,3’-cAMP and measured renal venous levels of
2’,3’-cAMP and 2’-AMP
Is the Metabolism of Endogenous 2’,3’-cAMP Altered in Kidneys from CNPase -/- Mice?
Isolated and perfused kidneys from CNPase +/+ vs -/- mice, administered metabolic poisons, dropped kidneys directly into liquid nitrogen,
extracted purines and measured total kidney levels of purines.
Is the Metabolism of 2’,3’-cAMP Altered in Kidneys from CNPase -/- Mice?
Isolated and perfused kidneys from CNPase +/+ vs -/- mice, administered metabolic poisons, dropped kidneys directly into liquid nitrogen,
extracted purines and measured total kidney levels of purines.
0 1 2 3 4 5 60
20
40
60
80
100
0 1 2 3 .4 5 6
0
20
40
60
80
100
2’,3’-cAMP
3’,5’-cAMP
CNPase -/-2’,3’-cAMP
3’,5’-cAMP
CNPase +/+
Rela
tive
Abun
danc
e
Retention Time (Minutes)
Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?
15 min
Induced AKI in CNPase +/+ versus CNPase -/- mice and examined renal function and structure 48 hours later.
Grenz et al., 2011 www.jove.com
Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?
Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?
Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?
Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?
Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?
B
C
A
ICAM
MCP1
Bottom Line:Knocking Out CNPase Attenuates
Acute Kidney Injury!!
BUT WHY??
CELL2’,3
’-cAMP
2’-AMP
ADO
mRNA
Ecto-2’,3’-PDEs
Ecto-2’/3’-NTs
TissueProtection
Extracellular 2’,3’-cAMP-Adenosine Pathway
2’,3’-cAMP
Transp
orter
RNases
mPTPs
Apotosis
Bad Stuff
CNPaseX2’-AMP + 3’-AMP
Mitohorm
esis
Does 2’-AMP Cause Bad Stuff?
Yes, 2’-AMP is a Potent RenalVasconstrictor!
2’-AMP is Renal Vasoconstrictor
2’-AMP Also Reduces GFR
Inhibition of Alkaline Phosphatase Induces AKI
CELL2’,3
’-cAMP
2’-AMP
ADO
mRNA
Tissue Protection
2’,3’-cAMP
Transp
orter
RNases
mPTPs
Renal Vasoconstriction
CNPase
Mitohorm
esis
Alkaline Phosphatase
CNPase
Inhibitor
Inhibitor o
f 2’-A
MP-
Induced Renal
Vasoco
nstricti
on
AlkalinePhosphatase
Opportunities for Treating/Preventing AKI
Induce
Mitohorm
esis
Alkaline Phosphatase for Sepsis-Induced AKI
Heemskerk et al.Critical Care Medicine 2009 Feb;37(2):417-423
Alkaline phosphatase treatment improves renal function in severe sepsis or septic shock patients.
Pickkers et al.Critical Care 2012 Jan 23;16(1):R14.
Alkaline phosphatase for treatment of sepsis-induced acute kidney injury: a prospective randomized double-blind placebo-controlled trial.
Future Directions?
What is the mechanism of 2’-AMP-induced renal vasoconstriction? Can we block 2’-AMP-induced renal vasoconstriction?
Would blockade of 2’-AMP-induced vasoconstriction prevent/treat AKI?
Can we develop a selective CNPase inhibitor?Would pharmacological inhibition of CNPase prevent/treat AKI?
Would administration of alkaline phosphatase prevent/treat AKI?
What happens to renal mitochondria when CNPase is blocked?Would inducing mitohormesis prevent AKI?