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0.1µM cGMP 1µM cGMP 10µM cGMP0
20
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CVH: Neurons inhibited by cGMP
Extracellular cyclic GMP (cGMP), the downstream mediator released in response to linaclotide-induced activation of guanylate cyclase-C (GC-C), reduces excitability of murine and human dorsal root ganglion neurons
BACKGROUND
ACKNOWLEDGEMENTS Supported by Ironwood Pharmaceuticals Inc, Forest
Laboratories and NHMRC Australia.
SUMMARY
REFERENCES
RESULTS
• Linaclotide, an investigational GC-C agonist,
improves abdominal pain and bowel symptoms in
humans with Irritable Bowel Syndrome with
Constipation (IBS-C)1.
Colonic DRG neurons from CVH mice displayed a significantly reduced rheobase, firing significantly more action potentials compared with healthy mice at baseline.
In a subpopulation of colonic DRG neurons, cGMP inhibited the neuronal excitability of putative nociceptors, significantly increasing their rheobase and reducing action potential discharge.
This effect was evident in both healthy and CVH DRG neurons, was most apparent in CVH DRG neurons, and occurred at concentrations as low as 100nM cGMP.
In human DRG neurons, cGMP induced an overall reduction in the number of cells responding to hypo-osmotic stimulation.
In addition in human DRG neurons cGMP caused, in a concentration-dependent manner, up to 60% inhibition of the Ca2+ influx induced by hypo-osmotic stimulation.
Mouse DRG studies
AIMS
CONCLUSIONS Exogenous cGMP directly decreases the
excitability of sensory DRG neurons isolated from both mice and humans.
These results complement our previous findings in mice, which demonstrated that cGMP inhibited the peripheral endings of nociceptors within the wall of the colon.
These current findings also provide further mechanistic insight into how linaclotide, through GC-C agonism and the release of cGMP from mucosal epithelial cells, reduces nociceptive signaling from the colon.
Colonic sensory neurons reside in the thoracolumbar (TL: T10-L1) and lumbosacral (LS: L6-S1) dorsal root ganglia (DRG), with afferents projecting via the splanchnic and pelvic nerves respectively. Five different subclasses of mechanosensitive afferent innervate the colorectum via these pathways. Modified from 3.
1) Castro et al., and Brierley 2013, Gastroenterology. 2) Brierley et al., Gastroenterology, 2004, Jul;127(1):166-78. 3) Brierley. Autonomic Neuroscience. 2010. Special Edition, 153, 1-2, 58-68. 4) Brierley. Current Opinion in Pharmacology. 2012. Special edition. 2012
Dec;12(6):632-40. 5) Brierley et al., Journal of Physiology 2011 Jul 15;589(Pt 14):3575-93. 6) de Araujo et al., Brierley & Alewood. Nature Communications.
2014;5:3165.
1,2 Joel Castro, 3 Grigori Y. Rychkov, 4 Andrea Ghetti, 1,2 Andrea M. Harrington, 5 Caroline Kurtz, 5 Ada Silos-Santiago and 1,2,3 Stuart M. Brierley 1 Visceral Pain Group, Discipline of Medicine, SAHMRI, 2Dept of Gastroenterology & Hepatology, Royal Adelaide Hospital. 3 Discipline of Physiology, University of Adelaide, AUSTRALIA. 4 Anabios Inc, USA. 5 Ironwood Pharmaceuticals Inc, USA.
• Linaclotide inhibits colonic nociceptors in vitro 1.
Exogenous cGMP causes greater inhibition of CVH colonic nociceptors
cGMP (µM)
Healthy nociceptors
n=19
* *
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0 1 50 250 1000
CVH nociceptors
n=19
*** ***
0
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0 1 50 250 1000
***
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cGMP (µM)
Exogenous cGMP mimics the effect of linaclotide by inhibiting colonic nociceptors
Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346
Linaclotide causes greater inhibition of colonic nociceptors in mice with CVH
Linaclotide (nM)
CVH nociceptors
Me
chan
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n=10
****** ***
0 1 30 100 300 1000
***
0
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Change from baseline: healthy vs. CVH
Linaclotide (nM)1 30 100 300 1000
-6
-3
0
-9
-12
-15Ch
ange
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HealthyCVH
***
******
*
***P<0.001
*P<0.05, ***P<0.001
Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346
• Cyclic GMP (cGMP) is a second messenger
produced in intestinal epithelial cells when
activated by guanylate-cyclase C (GC-C)
agonists 1.
In chronic visceral hypersensitivity (CVH) mice linaclotidehas greater efficacy in reducing nociceptive signaling in
response to noxious colorectal distension (CRD)CVH CRD
Linaclotide + CVH CRD
CVH, N=5.Linaclotide + CVH, N=4.
*** ****
0
5
10
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20
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K-I
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T10-T11 T11-T12 T13-L1
Spinal segment
*P<0.05. ***P<0.001
Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346
Phase III clinical trial of 805 IBS-C patients: Linaclotide significantly improves abdominal pain
Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346
• In vivo intra-colonic administration of linaclotide
also reduces nociceptive signalling from the colon 1.
• cGMP also inhibits colonic nociceptors in vitro
• We performed whole cell patch clamp recordings 5 in current clamp mode from retrogradely traced thoracolumbar (T10-L1) colonic DRG neurons.
• We compared the effect of exogenous extracellular cGMP (100nM-50μM) on the rheobase, or threshold for action potential firing, of DRG neurons from healthy C57BL/6 mice and mice with CVH, 28 days post-TNBS administration 1,6.
• We performed calcium-imaging studies and compared the effects of cGMP (10μM-300μM) on calcium (Ca2+) influx in response to hypo-osmotic stimuli of DRG neurons from healthy donors.
• Pain from the colon and rectum is signalled via
two distinct anatomical spinal afferent pathways 2.
Sacral
Spinal Cord
Lumbar
Thoracic
Key components of the GC-C/cGMP pathway
Abbreviations:GTP: Guanosine triphosphatePKGII: Protein kinase II.NHE3: Sodium/hydrogen exchanger 3.CFTR: Cystic fibrosis transmembrane conductance regulator.
Guanylin
cGMP transporters
Therefore, both linaclotide and exogenous extracellular cGMP inhibit the peripheral endings of colonic nociceptors, with greater efficacy during CVH 1.
However, the effects of exogenous cGMP on sensory neuron function remain to be determined in isolation.
We investigated the effects of exogenous extracellular cGMP on dorsal root ganglion (DRG)
neurons isolated from both mice and humans.
METHODS
Human DRG studies
Mouse DRG studies
Human DRG studies
Human DRG studies
Human DRG neurons were tested with a hypo-osmotic stimuli (70mM NaCl; 5mM KCl, 2mM CaCl2, 1mM
MgCl2, 10mM HEPES, 10mM glucose, pH 7.40, 165 mOsm) followed by capsaicin (200nM).
Neurons that did not respond to either stimuli were classified as “un-responsive’. Neurons responding to
the hypo-osmotic stimuli, but not capsaicin, were termed “mechano-responsive”. Neurons responding to
capsaicin only were termed “capsaicin-responsive”, whilst neurons responding to both stimuli were termed
“Mechano- and Capsaicin responsive”.
Hypo-osmotic stimuli and capsaicin reveal distinct
sub-populations of human DRG neurons
Responsiveness of human DRG neurons to hypo-
osmotic stimuli is dose-dependently decreased by
exogenous extracellular cGMP application
Exogenous extracellular cGMP application causes
greater inhibition in a select sub-population of
human DRG neurons
Human DRG neurons were tested with a hypo-osmotic stimuli (70mM NaCl; 5mM KCl, 2mM CaCl2, 1mM
MgCl2, 10mM HEPES, 10mM glucose, pH 7.40, 165 mOsm) followed by increasing doses (10µM-
300µM) of cGMP (MM-431343). Each dose of cGMP was applied for 2 minutes. After each dose of cGMP
neuronal responsiveness to the hypo-osmotic stimuli was re-tested.
Human DRG neurons were split into specific subpopulations based on their responsiveness to hypo-
osmotic stimuli and capsaicin. This revealed that mechano-responsive neurons were inhibited at lower
concentrations of exogenous extracellular cGMP.
Mouse colonic DRG studies TL colonic DRG neurons from CVH mice display
neuronal hyper-excitability
Healthy mice: Exogenous extracellular cGMP
application inhibits colonic DRG neurons
CVH mice: Exogenous extracellular cGMP application
causes potent inhibition of colonic DRG neurons
Healthy mice: A) A TL colonic DRG
neuron responding to 10pA
depolarizing stimuli in control bath
solution. The rheobase of this neuron
is160pA. B) In the same neuron a 2
min application of 10µM cGMP
increased the rheobase to 280pA. C)
Rheobase is maintained at 280pA
following 2 min 50µM cGMP.
D) Rheobase is partially normalized
after washout of cGMP. E) Group
data showing that cGMP increases
the rheobase of healthy colonic DRG
neurons. Data presented as the
change in rheobase from healthy
control solution baseline.
240 pA
0 pA 0pA
240pA
Healthy mouse colonic DRG neuron CVH mouse colonic DRG neuron
E)
A) CVH: Control solution
Rheobase: 90pA
0 pA
240 pA
B) CVH: 0.1μM cGMP
Rheobase: 140pA
C) CVH: 1μM cGMP
Rheobase: 140pA
D)
A) TL colonic DRG neurons from CVH mice have a significantly reduced rheobase (***P<0.001, Healthy: n=23
vs. CVH: n=13). B) CVH colonic DRG neurons fire more action potentials at 2x rheobase (**P<0.01, Healthy:
n=23 vs. CVH: n=13. C&D) Current clamp recordings of TL colonic DRG neurons in response to depolarizing
stimuli (10pA current steps) from C) Healthy and D) CVH mice.
D) C)
B) A)
CVH mice: A) TL colonic DRG
neuron responding to depolarizing
stimuli (10pA) in control bath solution.
The rheobase of this neuron is 90pA.
B) In the same neuron a 2 min
application of 0.1µM cGMP increases
the rheobase to 140pA. C) Rheobase
is maintained at 140pA following 1µM
cGMP. D) Group data showing that
cGMP dose-dependently increases
the rheobase of CVH colonic DRG
neurons. Data presented as change in
rheobase from CVH control solution
baseline.
Modified from 4.