Adrenergic Stimulation Mediates Visceral Hypersensitivity to Colorectal Distension Following Heterotypic Chronic Stress  John H. Winston, Guang–Yin Xu, Sushil K. Sarna  Gastroenterology  Volume 138, Issue 1, Pages 294-304.e3 (January 2010) DOI: 10.1053/j.gastro.2009.09.054 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 (A) HeCS significantly increased visceromoter to graded CRD (n = 14 rats; *P < .05) compared to prestress baseline responses 8 hours after the last stressor. No significant increase was observed at 24 hours and 7 days after HeCS. (B) Sham-stressed control rats showed no change in their visceromoter response at 8 and 24 hours, but significant differences were found between baseline and 7 days at 40, 50, 60, and 80 mmHg; *P < .05, n = 10 rats. Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Effects of HeCS and colonic resiniferatoxin (RTX) treatment on electrophysiological properties of 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocynine perchlorate (DiI)-labeled colon-specific thoracolumbar DRG neurons. Changes in electrophysiological properties of colon-specific DRG neurons from control (Ctr, n = 31 neurons), HeCS (n = 22 neurons), HeCS rats pretreated with RTX (n = 23 neurons), and control rats treated with RTX (n = 20 neurons). (A) Current clamp traces showing action potentials (AP) induced by current injection at rheobase. (B) Graph showing changes in rheobase. (C) Graph showing changes in resting membrane potential. (D) Current clamp traces showing the number of action potentials at 2× rheobase. (E) Number of AP induced by current injection at 2× rheobase. *P < .05, control vs HeCS, #P < .05 HeCS vs HeCS + RTX. Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 (A) HeCS significantly increased NGF in muscularis externa and mucosa/submucosa, which was blocked by inhibition of α1/α2 and β1/β2-adrenergic receptors prior to each daily stress session; n = 8 rats in each group, *P < .01, control (Ctr) vs HeCS and HeCS vs HeCS + adrenergic receptor antagonists. (B) Immunohistochemical staining for NGF (brown) in distal colon cross-sections from control and HeCS rats. Sections were counterstained with hematoxylin. (C) NGF antagonism by systemic administration of neutralizing antibody significantly reduced HeCS-induced increase in visceromoter response. HeCS, n = 4 rats, HeCS + NGF Ab, n = 5 rats, *P < 0.05 baseline vs HeCS; +P < .05 HeCS vs HeCS + NGF Ab. (D) Intrathecal administration of trkA antagonist k252A (n = 5) or antisense oligonucleotide (n = 3) suppressed HeCS-induced increase in visceromoter response. *P < .05 baseline vs HeCS, +P < .05, HeCS + mismatch (MM) oligonucleotide vs HeCS + NGF Ab. (E) Western blots showing the effects of intrathecal treatment with either trkA antisense (AS) or MM oligonucleotide on trkA receptor expression in thoracolumbar (TL), thoracic (Th), or lumbosacral (LS) DRG; n = 4; *P < .05 MM vs antisense oligonucleotides. Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Intrathecal treatment with trkA antagonist k252a blocked the HeCS-induced increase in excitability of colon-specific thoracolumbar DRG neurons (Ctr = control, n = 13 neurons from 5 rats; HeCS, n = 22 neurons from 5 rats; HeCS pretreated with resiniferatoxin, n = 23 neurons from 5 rats. (A) Rheobase. (B) Resting membrane potential. (C) Number of action potentials (APs) at 2× rheobase. *P < .05, control vs HeCS, #P < .05 HeCS vs HeCS + k252a. Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Electrophysiological properties of colon-specific thoracolumbar DRG neurons that were incubated for 24 hours with either high NGF (250 ng/mL) or low NGF (2.5 ng/mL) in vitro. Neurons incubated with high NGF showed a significant decline in resting membrane potential (A) and rheobase (B), *P < .05, low NGF vs high NGF. Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 (A) In vivo intraperitoneal administration of α1/α2- and β1/β2-adrenergic receptor antagonists blocked the HeCS-induced increase in the visceromoter response to graded CRD (n = 3). Rats subjected to HeCS were treated once daily before each stress session with a combination of phentolamine (2 mg/kg intraperitoneal [ip]) + and propranolol (2 mg/kg ip). In vitro incubation of muscularis externa/serosa (B) and mucosa/submucosa (C) for 24 hours with norepinephrine concentration-dependently increased expression of NGF; *P < .05, n = 6 strips (30 strips of each tissue type were prepared from distal colon of 4 rats [about 8 strips/rat] and evenly distributed among the experimental groups). Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 (A) HeCS did not increase the number of mast cells in the muscularis externa or mucosa/submucosa (n = 4 rats). (B) and (C) HeCS did not increase the spontaneous or ionomycin-induced release of tryptase from the muscularis externa or the mucosa/submucosa. Instead, it decreased the spontaneous release of tryptase in the muscularis externa; n = 4 rats; *P < .05. (D) HeCS did not increase MPO protein in the muscularis externa or mucosa/submucosa; n = 4 rats. (E) and (F) CRH1/CRH2 receptor antagonist astressin (AST) had no effect on the norepinephrine-induced expression of NGF in the muscularis externa/serosa, but it blocked this increase in mucosa/submucosa, *P < .05, n = 4 strips. Strips were incubated with 10 μM AST for 30 minutes before addition of norepinephrine (NE). Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 A schematic illustrating the proposed mechanism of HeCS-induced visceral hypersensitivity to CRD in relation to the well-established elements of the stress response. Step 1: Stress releases of CRH and angiotensin vasopressin from the paraventricular nucleus in the hypothalamus. CRH releases adrenocorticotropic hormone and other mediators from the pituitary. Step 2: CRH and arginine vasopressin stimulate the locus ceruleus/norepinephrine system. Step 3: The activation of the sympathetic preganglionic neurons releases norepinephrine/epinephrine in blood stream from the adrenal medulla. Step 4: Norepinephrine elevates the expression of NGF in colonic muscularis externa and mucosa/submucosa. Step 5: NGF complexes with trkA receptors and the complex transports retrograde to the thoracolumbar DRG. Step 6: NGF/trkA complex sensitizes colon-specific neurons by modulating the expression and characteristics of ion channels Step 7: The amplification of afferent signal in response to CRD is perceived as abdominal pain/discomfort. Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 The effect of heterotypic chronic stress (HeCS) on the expression of nerve growth factor (NGF) (13 kD band) in the muscularis externa and mucosa/submucosa was confirmed by Western blotting. Muscularis externa and mucosa/submucosa tissue samples were obtained from 3 control (Ctr) rats and 3 rats subjected to HeCS protocol. HeCS significantly increased the expression of NGF in both tissues. *P < .05, n = 4 (3 shown). Gastroenterology 2010 138, 294-304.e3DOI: (10.1053/j.gastro.2009.09.054) Copyright © 2010 AGA Institute Terms and Conditions