Skip to main content
NCBI home page
American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2019 Nov 4;318(1):G23–G33. doi: 10.1152/ajpgi.00247.2019

BDNF augments rat internal anal sphincter smooth muscle tone via RhoA/ROCK signaling and nonadrenergic noncholinergic relaxation via increased NO release

Arjun Singh 1, Ipsita Mohanty 2, Jagmohan Singh 3, Satish Rattan 1,✉
PMCID: PMC6985850  PMID: 31682160

graphic file with name zh3001207706r001.jpg

Open in a new tab

Keywords: NANC relaxation, neurotrophic factor, RhoA/ROCK, smooth muscle tone

Abstract

Presently, there are no studies examining the neuromodulatory effects of brain-derived neurotropic factor (BDNF) on the basal internal anal sphincter (IAS) tone and nonadrenergic noncholinergic (NANC) relaxation. To examine this, we determined the neuromuscular effects of BDNF on basal IAS smooth muscle tone and the smooth muscle cells (SMCs) and the effects of NANC nerve stimulation before and after high-affinity receptor tyrosine kinase receptor B (TrkB) antagonist K252a. We also investigated the mechanisms underlying BDNF-augmented increase in the IAS tone and NANC relaxation. We found that BDNF-increased IAS tone and SMC contractility were TTX resistant and attenuated by K252a. TrkB-specific agonist 7,8-dihydroxyflavone, similar to BDNF, also produced a concentration-dependent increase in the basal tone, whereas TrkB inhibitors K252a and ANA-12 produced a decrease in the tone. In addition, BDNF produced leftward shifts in the concentration-response curves with U46619 and ANG II (but not with bethanechol and K+ depolarization), and these shifts were reversed by K252a. Effects of Y27632 and Western blot data indicated that the BDNF-induced increase in IAS tone was mediated via RhoA/ROCK. BDNF-augmented NANC relaxation by electrical field stimulation was found to be mediated via the nitric oxide (NO)/soluble guanylate cyclase (sGC) pathway rather than via increased sensitivity to NO. In conclusion, the net effect of BDNF was that it caused an increase in the basal IAS tone via RhoA/ROCK signaling. BDNF also augmented NANC relaxation via NO/sGC. These findings may have relevance to the role of BDNF in the pathophysiology and therapeutic targeting of the IAS-associated rectoanal motility disorders.

NEW & NOTEWORTHY These studies for the first time to our knowledge demonstrate that increased levels of brain-derived neurotrophic factor (BDNF; conceivably released from smooth muscle cells and/or the enteric neurons) has two major effects. First, BDNF augments the internal anal sphincter (IAS) tone via tyrosine kinase receptor B/thromboxane A2-receptor, angiotensin II receptor type 1/RhoA/ROCK signaling; and second, it increases nonadrenergic noncholinergic relaxation via nitric oxide/soluble guanylate cyclase. These studies may have relevance in therapeutic targeting in the anorectal motility disorders associated with the IAS.

INTRODUCTION

Brain-derived neurotrophic factor (BDNF), as the name denotes, was initially demonstrated to be present and active in the brain and the rest of the central nervous system (CNS) (23, 52). BDNF is a part of the neurotrophin family of growth factors that also includes NGF and neurotensins (NT)-3 and NT-4/5 (9, 34). These factors are known to regulate neuronal proliferation, differentiation, survival, migration, dendritic arborization, synaptogenesis, and synaptic plasticity in the CNS (53).

More recently, besides its role in the CNS, BDNF has also been shown to be present in a number of peripheral organs, including different smooth muscle systems (1, 8, 21). A number of studies have now shown that BDNF is generated within the smooth muscle cells (SMCs) of different organs, such as airways and the gastrointestinal tract (1, 8, 21, 22). In the gastrointestinal tract, BDNF appears to augment peristaltic reflex via neuronal and nonneuronal actions (1, 22, 29). BDNF works via activation of high-affinity receptors tyrosine receptor kinase B (TrkB) and low-affinity p75 NT receptor (p75NTR) (8, 26). This BDNF/TrkB signaling involves a number of complex pathways, including Ca2+/myosin light chain kinase and RhoA/ROCK pathways (7, 8, 19, 52).

Little else is known about the effect and role of BDNF in the gastrointestinal tract, particularly its potential effect on the basal tone of the internal anal sphincter (IAS) and nonadrenergic noncholinergic (NANC) relaxation. The IAS performs two important functions: 1) rectoanal continence via the myogenic basal tone and 2) the rectoanal inhibitory reflex (RAIR, also known as defecation reflex) via the NANC inhibitory neurotransmitter, primarily nitric oxide (NO) (43, 54). A compromise in the basal IAS tone leads to rectoanal incontinence, and a compromise in the RAIR may lead to constipation (5, 6, 36, 37). Basal myogenic IAS tone initiated by the Ca2+/myosin light chain kinase pathway is maintained molecularly by RhoA/ROCK (38). Despite significant advances in our current understanding of the basic underlying mechanisms for the basal tone and the nature of inhibitory neurotransmitters, targeted therapy for the debilitating anorectal disorders related to the IAS function is not available.

Therefore, the purpose of the present investigation was to determine the excitatory effect in the basal tone and the neuromodulatory effect of BDNF in the RAIR. To mimic RAIR, for in vitro studies, we used electrical field stimulation that works via the release of the NANC inhibitory neurotransmitter NO (40, 43). Our studies focused on the acute effects of BDNF via TrkB using exogenous BDNF and the TrkB agonists 7,8-dihydroxyflavone (7,8-DHF), K252a, and ANA-12 (2, 3, 33, 56). We found that BDNF increased the basal and agonist-stimulated IAS tone via TrkB/thromboxane A2-receptor (TXA2-R)/angiotensin II receptor type 1 (AT1-R)/RhoA/ROCK signaling, and RAIR-induced IAS relaxation via NO/soluble guanylate cyclase (sGC) pathway.

MATERIALS AND METHODS

Animals and IAS Tissues Preparation

Male adult Sprague-Dawley rats (8–10 mo old, weighing 250–350 g) were utilized in the present studies. The Institutional Animal Care and Use Committee of Thomas Jefferson University approved all experimental protocols, and studies were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.

After decapitation of isoflurane-anesthetized rats, the anorectal tissue was carefully removed and transferred to oxygenated (95% O2 + 5% CO2) ice-cold (4°C) Krebs physiological solution (KPS) of following composition: 118.07 mM NaCl, 4.69 mM KCl, 2.52 mM CaCl2, 1.16 mM MgSO4, 1.01 mM NaH2PO4, 25 mM NaHCO3, and 11.1 mM glucose. The smooth muscle strips (∼1 × 10 mm) from the circular smooth muscle layer of the IAS were isolated and prepared for the isometric tension experiments as previously described (32).

Isometric Tension Measurement

The IAS SM strips prepared above were mounted in 2-mL organ baths (Radnoti, Monrovia, CA) containing oxygenated (95% CO2 + 5% O2) KPS at 37°C and employed for the isometric contraction studies as described previously (32). The basal tone in the IAS and its changes were monitored using force transducers (FORT10; World Precision Instruments, Sarasota, FL) and recorded using Chart v4.1.2 via a PowerLab/8SP data acquisition system (ADInstruments, Colorado Springs, CO). The SM strips were stretched to a tension of 1 g, followed by an equilibration period of 60 min to develop the spontaneously active basal IAS tone. The basal IAS tone and its changes were calculated as percent maximal decrease (with 0 Ca2+) and percent maximal increase in the IAS tone (recorded following 10−4 M bethanechol), as described previously (47).

Electrical Field Stimulation

Electrical field stimulation (EFS) was delivered using S11Grass stimulator (Grass Instruments, Quincy, MA) with optimal stimulus parameters (10 V, 0.5-ms pulse duration, 4-s train, at 0.5–20 Hz) under NANC conditions (in the presence of 1 μM guanethidine and 1 μM atropine) (40, 42).

SMC Contractility and Relaxation Studies

Isolation of the SMCs.

SMCs were isolated from the IAS per our previous studies (32, 50). Briefly, the circular smooth muscle tissues were cut into 1-mm cubes and incubated in oxygenated KPS containing 0.1% collagenase type 2, and 0.01% soybean trypsin inhibitor at 37°C for 3 h. After this step, the cell suspension was centrifuged and filtered through Nitex mesh (500 μm). The cell suspension was then centrifuged at 350 g for 5 min and plated on collagen-coated plates in DMEM (Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, 1% antimycotic antibiotic solution 100×, and 50 μg/mL sodium ascorbate) in a 75-cm2 cell culture flask at 37°C and 5% CO2 incubator with regulated humidity.

Measurement of changes in SMC lengths (morphometric analysis).

The length of freshly isolated SMCs (in the basal state and following different agents) were measured by scanning micrometry under phase-contrast microscopy (Nikon Eclipse-TE2000-S, Nikon, Tokyo, Japan), as described previously (18, 49). Freshly isolated SMCs were resuspended in oxygenated KPS (at 37°C) at a cell density of 3 × 104 cells/mL in 30-μL aliquots. Each IAS SMC aliquot was treated for 5 min with different concentrations of U46619, KCl, isoproterenol, VIP, or sodium nitroprusside (SNP) (before and after 15 min of pretreatment with either BDNF, K252a, or BDNF + K252a) were fixed with final concentration of 0.1% acrolein, and the glass slides were covered with glass coverslips. Phase-contrast images of the SMCs (50 cells each from at least 3 animals) were stored digitally and the cell lengths were measured by using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD). Shortening of the SMCs in each category of experiments was calculated as percentile of basal cell lengths.

Nitric Oxide Measurement Studies

For NO measurements from the muscle bath perfusates, we followed the previously established protocols from our laboratory with certain modifications (4, 11, 12, 40). Herein, we used chemiluminescence NO detector (Zysense 280i Nitric Oxide Analyzer, Zysense Instruments, Fredrick, CO) coupled with ozone-chemiluminescence technology using liquid analyze software (16). After calibration, 50 μL of each sample (collected before and after different EFS Hz, in the absence and presence of either BDNF, K252a, or BDNF + K252a) was injected to measure the NO levels. In these experiments, KPS was used as a negative control, and different concentrations of freshly prepared sodium nitrite [NaNO2] were used for the standard curve. All samples were analyzed in a dark and O2-free environment.

Western Blot Analysis

The IAS SMCs and tissue lysates were prepared and subjected to Western blot analysis for RhoA and ROCK2, as described previously (48, 51). Aliquots of these lysates prepared as control, BDNF, and BDNF + K252a groups were analyzed for ROCK2; and BDNF, BDNF + losartan, and BDNF + SQ 29,548 groups were analyzed for RhoA and ROCK2. (BDNF was used in 1 nM, and other agents were used in 100 nM concentration). For these experiments, GAPDH served as a positive control (32, 47, 49).

Briefly, total protein from each sample was separated by using SDS-polyacrylamide gel (7.5% gel for ROCK2, and 15% gel for RhoA) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) through iBlot dry blotting system (Invitrogen, Carlsbad, CA) at room temperature. Protein concentrations were analyzed by using the bicinchoninic acid protein method (Pierce Biotechnology, Rockford, IL). The membranes were subjected to immunoblot analysis using specific primary antibodies (1:1,000 dilution of RhoA, ROCK2) and IRDye680-conjugated and IRDye800-conjugated secondary antibodies (bovine anti-rabbit 1:500 dilution for RhoA/ROCK2) from LI-COR (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature in dark. These membranes were then scanned using a LI-COR infrared scanner, and the integrated optical densities (as ratios of GAPDH) were determined using ImageJ software (NIH, Bethesda, MD).

Drugs and Chemicals

Angiotensin II, ATP, bethanechol chloride, glacial acetic acid, isoproterenol, potassium iodide, SNP, substance P, TTX, VIP, and Y27632 were purchased from Sigma Aldrich (St. Louis, MO). ANA-12, BDNF, 7,8-DHF, hemoglobin, K252a, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and U46619 were obtained from Tocris Biosciences (Minneapolis, MN). KCl, and sodium nitrite were purchased from Fisher Scientific (Pittsburgh, PA). RhoA, ROCK2, and GAPDH antibodies were purchased from Invitrogen (Waltham, MA). SQ 29,548 and TNF-α were purchased from Cayman Chemical (Ann Arbor, MI), and BD Biosciences (San Jose, CA), respectively. Losartan was a gift from Merck (Rahway, NJ).

Statistical Analysis

Data were presented as the means ± SE of multiple experiments, and P value < 0.05 was considered statistically significant. All data were analyzed and graphed using GraphPad Prism 5.0 (San Diego, CA). For comparisons between two groups, Student’s t test was performed. The concentration-response curves were fitted by nonlinear regression, and comparison was made using one-way ANOVA.

RESULTS

Schematics of the experimental protocols using smooth muscle strips to determine changes in the IAS tone in the basal state, agonist-stimulated state, and following EFS before and after different maneuvers appear in Fig. 1. These data were collected during controls, followed by the treatment with BDNF before and after the TrkB antagonist K252a were added in the concentrations, and for the duration, as shown in Fig. 1. These studies were followed by the SMC experiments and then by the mechanistic studies for the BDNF-induced increase in the basal tone and NANC relaxation. Not shown here, in some studies, we also examined the effects of TrkB agonist 7,8-DHF and antagonist ANA-12.

Fig. 1.

Fig. 1.

Open in a new tab

A simplified flow chart of experimental protocols for examining changes in the internal anal sphincter tone in the basal state following different agonists and electrical field stimulation (EFS) before and after pretreatment with brain-derived neurotrophic factor (BDNF), K252a, and BDNF + K252a. The washout effect was ascertained by the return of the basal tone and EFS and agonists’ responses to the prewashout levels. In addition, we examined the effects of single exposures to BDNF, K252a, and BDNF + K252a. Concentrations of BDNF, K252a, and their incubation times are shown in the flow chart.

Effect of BDNF in the Basal IAS Tone Before and After TrkB-Selective Antagonist K252a

BDNF produced a concentration-dependent and time-dependent increase in the IAS tone, with the maximal effect occurring with 1 nM and plateauing at 30–40 min. This significant increase in the tone (as compared with control) was maintained for up to 3 h (*P < 0.05; n = 12 in 6 animals; Fig. 2A). Therefore, for the subsequent BDNF studies, we used 1 nM and 1 h of incubation. The BDNF-induced increase in the basal tone was not significantly modified by TTX (1 μM; P > 0.05; n = 4 in 4 animals; Fig. 2B).

Fig. 2.

Fig. 2.

Open in a new tab

A: data show that brain-derived neurotrophic factor (BDNF) causes a significant and concentration-dependent increase in the internal anal sphincter (IAS) tone (*P < 0.05) which is maintained for 3 h, and that 1 nM BDNF produces the maximal effect. B: data show that TTX has no significant effect on BDNF (1 nM; 1 h incubation)-increased IAS tone (*P > 0.05). All data here and elsewhere are represented as the means ± SE.

Interestingly, BDNF mimetic and a small molecule TrkB agonist, 7,8-DHF, produced similar increases in the IAS tone, as did the BDNF, although with considerably less potency (*P < 0.05; n = 6 in 4 animals; Fig. 3A).

Fig. 3.

Fig. 3.

Open in a new tab

A: time course of the effects of brain-derived neurotrophic factor (BDNF) and 7,8-dehydroxyflavone (DHF) showing a significant (*P < 0.05) increase in the internal anal sphincter (IAS) tone, wherein BDNF is more potent than 7,8-DHF. B: data show that K252a as well as tyrosine kinase receptor B-selective inhibitor ANA-12 produce a significant (*P < 0.05) decrease in the IAS tone, K252a being several times more potent than ANA-12. C: BDNF levels are significantly higher in the IAS vs. rectal smooth muscle (RSM) in the basal state and following stimuli: ATP, TNF-α, and substance P (Sub P) (*P < 0.05).

Of equal importance, TrkB antagonists K252a and ANA-12 produced significant decreases in the IAS tone (*P < 0.05; n = 6 in 4 animals; Fig. 3B), however, ANA-12 was considerably less potent.

BDNF Release in the IAS Versus Rectal Smooth Muscles in the Basal and Stimulated States

To determine the specific relevance of BDNF in the IAS, we compared the release of BDNF in the IAS to its release in the adjoining rectal smooth muscle perfusates in the basal state and following stimulation with BDNF releasers ATP, TNF-α, and substance P. For these studies, we used rat BDNF ELISA Kit (cat. no. ERBDNF, Invitrogen) according to the manufacturer’s instructions. The analytical sensitivity was 12 and the assay range was 12.29–3,000 pg/mL. The assay diluent served as a control (0 pg/mL) for background subtraction to construct a standard curve. Data showed significantly higher levels of BDNF in the IAS versus rectal smooth muscle perfusates in the basal state as well as following different stimuli (*P < 0.05; n = 4 in 4 animals; Fig. 3C).

Effect of BDNF, Before and After K252a, on the Agonist-Induced Increase in the IAS Tone Produced by U46619, ANG II, Bethanechol, and KCl

These experiments revealed that BDNF produced a significant increase in the contractile effects of U46619 and ANG II (*P < 0.05; n = 4; Fig. 4, A and B), as shown by the significant leftward shifts in their control concentration-response curves (CRCs). In contrast, however, the effects of bethanechol and KCl CRCs were not significantly affected (P > 0.05; n = 8 in 4 animals; Fig. 4, C and D). In these experiments, the TrkB antagonist by itself had no significant effects on the CRCs of any of these agonists.

Fig. 4.

Fig. 4.

Open in a new tab

Data show that brain-derived neurotrophic factor (BDNF) causes a significant augmentation of increase in the internal anal sphincter tone by U46619 (A) and ANG II (B) (*P < 0.05) but not those by bethanechol (beth) (C) and KCl (D) (P > 0.05). Data further show that the BDNF receptor tyrosine kinase receptor B antagonist K252a blocks these augmentative effects, whereas K252a by itself has no significant effects.

Effect of BDNF on IAS Relaxation Caused by NANC Nerve Stimulation by Electrical Field Stimulation Before and After K252a

Using appropriate stimulus parameters as explained in materials and methods, electrical field stimulation (EFS) produced frequency-dependent (0.5–10 Hz) relaxation in the IAS that was significantly (*P < 0.05; n = 8 in 4 animals; Fig. 5A) increased when the experiments were repeated in the presence of BDNF. K252a attenuated this BDNF-mediated increase in the EFS-induced IAS relaxation.

Fig. 5.

Fig. 5.

Open in a new tab

A: brain-derived neurotrophic factor (BDNF) produces a significant increase in the internal anal sphincter relaxation caused by electrical field stimulation (EFS) (shown by the leftward shift in the EFS response curve) (*P < 0.05) is attenuated by K252a. B: bar graph data provides further details following 10 Hz of EFS. C: tracings on the right are the examples of the typical recordings of such experiments.

Figure 5B illustrates the data with 10 Hz of EFS, showing a significant increase (*P < 0.05; n = 8) in the IAS relaxation by BDNF was attenuated by K252a. In these experiments, K252a by itself had no significant effect on the EFS responses (P > 0.05). Figure 5C provides typical tracings of such experiments.

Effect of BDNF on Agonist-Induced IAS Relaxation

We carried out a series of experiments to determine whether BDNF-augmented NANC nerve stimulation resulted from increased sensitization of the relaxant responses to the released inhibitory neurotransmitter. These data revealed that, unlike the contractile effects of U46619 and ANG II, the inhibitory effects of any of the neurohumoral agonists (isoproterenol, VIP, and NO donor SNP) were not significantly modified by BDNF (P > 0.05; n = 8 in 4 animals; Fig. 6, A–C).

Fig. 6.

Fig. 6.

Open in a new tab

Data show that the relaxation produced by isoproterenol (A), VIP (B), and sodium nitroprusside (SNP) (C) is not significantly affected by either brain-derived neurotrophic factor (BDNF), K252a, or BDNF + K252a (P > 0.05).

To determine the direct effects of contractile and relaxant agonists and the influence of BDNF at the SMCs, we performed a series of experiments in the freshly isolated IAS SMCs as follows.

Effect of BDNF on the Length of the Basal IAS SMCs and on the Contractility of Agonist-Induced SMC

The addition of BDNF (1 nM) produced a significant decrease in the length of the IAS SMCs (*P < 0.05; Fig. 7, A and B) from basal cell lengths of 42.6 ± 1.2 to 29.5 ± 1.0 μm. This BDNF-induced shortening of the SMCs was attenuated by K252a (100 nM).

Fig. 7.

Fig. 7.

Open in a new tab

Data show absolute (A) and percent (B) changes in the smooth muscle cell (SMC) lengths in control, and following brain-derived neurotrophic factor (BDNF), K252a, and BDNF + K252a. These data show that BDNF causes significant contraction of the SMCs, which is attenuated by K252a, which by itself may cause relaxation (*P < 0.05). Additionally, BDNF significantly (*P < 0.05) augments the SMC contraction caused by U46619, which is attenuated by K252a (C), without modifying the effects of KCl (D).

BDNF also significantly augmented (*P < 0.05; Fig. 7C) the U46619-induced shortening of the SMCs, which was attenuated by K252a. However, the effects of KCl were not significantly modified by any of the maneuvers (P > 0.05; Fig. 7D).

Effect of BDNF on Agonist-Induced SMC Relaxation

Treatment with either BDNF, K252a, or BDNF + K252a did not significantly alter the effects of any of the relaxant agonists, isoproterenol, VIP, or SNP (P > 0.05; n = 8 in 4 animals; Fig. 8, A–C).

Fig. 8.

Fig. 8.

Open in a new tab

Consistent with the smooth muscle data, experiments with the smooth muscle cells (SMCs) show that relaxation by isoproterenol (A), VIP (B), and sodium nitroprusside (SNP) (C) are not significantly (P > 0.05) modified by any of the pretreatments: brain-derived neurotrophic factor (BDNF), K252a, or BDNF + K252a.

Effect of RhoA/ROCK Inhibitor Y27632 on Increase in the IAS Smooth Muscle Tone Caused by U46619

To determine the mechanism of action of BDNF, we obtained a series of data on the basal tonic state of the IAS after contractile and relaxant agonists and following NANC relaxation.

These data showed that BDNF caused a significant increase (*P < 0.05; n = 8 in 4 animals; Fig. 9A) in the control responses of U46619 as reflected by the leftward shift in the control U46619 CRC. These augmented responses of U46619 were significantly inhibited by Y27632 (1 μM; **P < 0.05; n = 8 in 4 animals; Fig. 9A). Interestingly, this inhibition of BDNF induced augmentation of U46619 responses by Y27632 was similar to that with K252a (100 nM).

Fig. 9.

Fig. 9.

Open in a new tab

A: Y27632 significantly (**P < 0.05) attenuates the augmentative effect of brain-derived neurotrophic factor (BDNF) on U46619 in a manner similar to that with K252a (**P < 0.05). B and C: BDNF causes a significant (*P < 0.05) increase in the expression of ROCK2, which is attenuated by K252a. D and E: BDNF causes a significant (*P < 0.05) increase in the expressions of RhoA/ROCK2, which are attenuated by thromboxane A2-receptor and angiotensin II receptor type 1 antagonists SQ 29,548 and losartan (LOS), respectively.

Effect of BDNF on TrkB/RhoA/ROCK2 Expression

Data in the IAS SMCs using Western blot analysis showed that BDNF caused a significant increase in the protein expression of ROCK2 (*P < 0.05; Fig. 9, B and C), and this increase was attenuated by TrkB antagonist K252a. Data further showed that control increases in the expressions of ROCK2 and RhoA by BDNF were significantly inhibited by losartan and SQ 29,548 (antagonists of AT1-R and TXA2-R, respectively; *P < 0.05; n = 4 in 4 animals; Fig. 9, D and E).

Collectively, the above data suggest that BDNF partially activates AT1-R and TXA2-R, which in turn activate RhoA/ROCK, leading to an increase in the IAS tone.

Effect of BDNF on NANC Nerve Stimulation-Induced NO Release

We performed these experiments to determine the role of NO release in the BNDF-induced augmentation of NANC relaxation. After standardization of NO measurements (Fig. 10A), data revealed that BDNF produced a significant increase (*P < 0.05; n = 6 in 4 animals; Fig. 10B) in the release of NO in the muscle bath perfusates taken immediately following 10 Hz EFS. K252a blocked this increased release in NO. Similar data (not shown) were obtained following the use of 5 Hz EFS.

Fig. 10.

Fig. 10.

Open in a new tab

Effect of brain-derived neurotrophic factor (BDNF) on increase in nitric oxide (NO) release in the internal anal sphincter following electrical field stimulation. A: the standard curve for the measurements of NO using present method. B: BDNF causes a significant increase (*P < 0.05) in the nonadrenergic noncholinergic nerve stimulation-induced release of NO, which is attenuated by K252a (**P < 0.05), whereas K252a by itself has no significant effect (P > 0.05).

Influence of NO Scavenger Hemoglobin, and sGC Inhibitor ODQ on BDNF-Augmented NANC Relaxation in the IAS

Hemoglobin (10 μM) as well as ODQ (1 μM) significantly attenuated BDNF-augmented NANC relaxation in the IAS (*P < 0.05; n = 6 in 4 animals; Fig. 11, A and B). Collectively, these data along with the NO release studies established the role of the NO/sGC pathway for the BDNF-induced augmentation of the NANC relaxation.

Fig. 11.

Fig. 11.

Open in a new tab

Effect of hemoglobin and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on nonadrenergic noncholinergic (NANC) relaxation in the internal anal sphincter. Data show that both hemoglobin (Hb) (A) and ODQ (B) significantly attenuate the brain-derived neurotrophic factor (BDNF)-induced increase in the NANC relaxation (*P < 0.05). EFS, electrical field stimulation.

DISCUSSION

Two major findings of our studies are outlined in Fig. 12. First, we found that BDNF increases the IAS tone via RhoA/ROCK involving TrkB/TXA2-R and TrkB/AT1-R signaling within the SMCs. Second, we found that BDNF augments NANC relaxation via NO/sGC.

Fig. 12.

Fig. 12.

Open in a new tab

Hypothetical model depicting role and mechanism of actions of brain-derived neurotrophic factor (BDNF) in the internal anal sphincter (IAS). Data suggest that BDNF (conceivably from different sources, including the smooth muscle cells (SMCs) and the enteric neurons) has two major effects: 1) an increase in the IAS tone via activation of tyrosine kinase receptor B (TrkB)/thromboxane A2-receptor (TXA2-R) and angiotensin II receptor type 1 (AT1-R)/RhoA/ROCK and 2) an increase in nonadrenergic noncholinergic (NANC) relaxation via nitric oxide (NO)/soluble guanylate cyclase (sGC). The net effect of BDNF in the IAS is an increase in the basal tone.

Our results suggest that BDNF produces a potent and sustained increase in the basal IAS tone by its action at the SMCs via activation of the BDNF receptor TrkB. The BDNF-induced increase in the IAS tone is selectively blocked by the TrkB antagonist K252a and not by the neurotoxin TTX. In addition, BDNF augments an agonist-stimulated increase in the tone via TrkB activation, selectively reversible by K252a. This selectivity was determined by attenuation of the contractile effect of BDNF on the basal tone and of TXA2-R agonist U46619 and AT1-R agonist ANG II, not of M3-R agonist bethanechol and K+ depolarization. Additionally, we found that BDNF produces contraction of the IAS SMCs in the basal as well as stimulated state (selectively by U46619, and not by K+ depolarization). In further support of the selectivity of the effect of BDNF and K252a, the TrkB antagonists, in contrast with their effects on the basal tone and on U46619 and ANG II, BDNF and K252a do not modify the relaxant effects of isoproterenol, VIP, and NO donator SNP in either the IAS smooth muscle strips or in the SMCs. This lack of effect of BDNF and K252a on the relaxation by NO in the IAS has further ramifications on the mechanism of action of BDNF in the NANC relaxation, as explained later.

Our data further showed that BDNF-induced SMC contractility involves TrkB/RhoA/ROCK signaling with the mediation of TXA2-R and AT1-R activation. Evidently, RhoA/ROCK inhibitor Y27632 inhibits BDNF-induced augmentation of U46619 responses in the IAS similar to that with K252a. BDNF increases the expression of ROCK2. Moreover, antagonism of TXA2-R (by SQ 29,548) and AT1-R (by losartan) attenuated the increase in RhoA/ROCK activation by BDNF. These data are significant in light of previously published studies showing that activation of TXA2-R and AT1-R via RhoA/ROCK are crucial in the genesis of basal IAS tone in animals as well as in humans (14, 15, 32, 38, 39, 41, 44).

The physiological significance of these studies in relation to the basal IAS tone is evident from significant increase in the IAS tone following not only BDNF but also 7,8-DHF, a selective TrkB agonist. In addition, the levels of BDNF in the muscle bath perfusate in the basal state and, following the stimulants (ATP, TNF-α, and substance P), were significantly higher in the IAS versus the adjoining smooth muscle of rectum. Furthermore, selective inhibitors of BDNF (K252a and ANA-12) produced significant decreases in the IAS tone.

The other important finding of the present studies was that BDNF augments NANC relaxation in the IAS. We tested two main hypotheses for the mechanism underlying this. First, we examined the role of an increase in the sensitization to the effect of the inhibitory neurotransmitter NO. Then, we tested the involvement of NO/sGC. The first hypothesis was ruled out by the lack of augmentative effect of BDNF on the IAS relaxation in response to exogenous NO. The second hypothesis in favor of NO/sGC was bolstered by the increased NO release and by inhibition of BDNF-augmented NANC relaxation of the IAS by hemoglobin and ODQ.

Presently, the exact source of BDNF in the IAS is not known. However, data from different laboratories suggest that high levels of BDNF in the end organs of different smooth muscle systems may be of multiple origins, such as the CNS, distant or local neuroendocrine cells, or the SMCs (1, 8, 21, 22). Regardless of the source, present data demonstrate that BDNF has two important sites of actions in the IAS: the SMCs and the NANC neurons.

Our findings generally agree with the previously published data showing that BDNF produces smooth muscle contraction and may be involved in the mediation and augmentation of gastrointestinal peristaltic reflex and motility (1, 22, 29). Most of these effects have been shown to occur via the increased release of ACh, 5-HT, and CGRP. Gastrointestinal peristalsis may occur via the activation of sensory neurons associated in turn with the descending inhibition via the release of NO (45).

Since one of the major components of rectoanal incontinence is a decrease in the IAS tone, it is possible that in some patients (including the aging population), BDNF/TrkB signaling is underexpressed and could be rectified by activation. Conversely, patients with Hirschsprung disease (HPD) (10, 24, 27, 31, 55) with an impairment in the RAIR relaxation integral may benefit from BDNF-augmented NANC relaxation. BDNF/TrkB are abundantly expressed in the enteric neural crest cells and in the enteric nervous system (28, 30, 53). In addition, BDNF is known to promote neuronal survival, growth, and differentiation in the functioning of the mature enteric nervous system (13, 22, 45, 52, 53). Interestingly, the segment of the distal intestine affected with HPD shows significant alteration in the BDNF/TrkB expression (25, 46). However, a direct link between HPD and BDNF remains to be established.

In summary, present studies examining short-term effects of BDNF in the IAS suggest that it augments basal tone via GPCR linked to RhoA/ROCK and enhances the NANC relaxation via NO/sGC signaling. These studies did not examine the long-term effects of BDNF and the contribution of other receptors, such as p75NTR, TrkA, and TrkC (17, 20, 25, 35). These findings have important pathophysiological and therapeutic implications in the IAS-associated rectoanal motility disorders.

GRANTS

This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-035385 and an institutional grant from Thomas Jefferson University.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.S., I.M., J.S., and S.R. conceived and designed research; A.S., I.M., and J.S. performed experiments; A.S., I.M., and J.S. analyzed data; A.S., I.M., J.S., and S.R. interpreted results of experiments; A.S., I.M., and J.S. prepared figures; A.S., I.M., J.S., and S.R. drafted manuscript; A.S., I.M., J.S., and S.R. edited and revised manuscript; A.S., I.M., J.S., and S.R. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors thank Jennifer Wilson for critical proof reading and valuable editorial suggestions.

REFERENCES

  • 1.Al-Qudah M, Alkahtani R, Akbarali HI, Murthy KS, Grider JR. Stimulation of synthesis and release of brain-derived neurotropic factor from intestinal smooth muscle cells by substance P and pituitary adenylate cyclase-activating peptide. Neurogastroenterol Motil 27: 1162–1174, 2015. doi: 10.1111/nmo.12604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Al-Qudah M, Anderson CD, Mahavadi S, Bradley ZL, Akbarali HI, Murthy KS, Grider JR. Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C. Am J Physiol Gastrointest Liver Physiol 306: G328–G337, 2014. doi: 10.1152/ajpgi.00203.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Azogu I, Plamondon H. Inhibition of TrkB at the nucleus accumbens, using ANA-12, regulates basal and stress-induced orexin A expression within the mesolimbic system and affects anxiety, sociability and motivation. Neuropharmacology 125: 129–145, 2017. doi: 10.1016/j.neuropharm.2017.07.008. [DOI] [PubMed] [Google Scholar]
  • 4.Banwait KS, Rattan S. Role of nitric oxide in β3-adrenoceptor activation on basal tone of internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 285: G547–G555, 2003. doi: 10.1152/ajpgi.00545.2002. [DOI] [PubMed] [Google Scholar]
  • 5.Bharucha AE, Dunivan G, Goode PS, Lukacz ES, Markland AD, Matthews CA, Mott L, Rogers RG, Zinsmeister AR, Whitehead WE, Rao SS, Hamilton FA. Epidemiology, pathophysiology, and classification of fecal incontinence: state of the science summary for the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) workshop. Am J Gastroenterol 110: 127–136, 2015. doi: 10.1038/ajg.2014.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Bharucha AE, Rao SS. An update on anorectal disorders for gastroenterologists. Gastroenterology 146: 37–45.e2, 2014. doi: 10.1053/j.gastro.2013.10.062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bothwell M. Recent advances in understanding neurotrophin signaling. F1000 Res 5: 1885, 2016. doi: 10.12688/f1000research.8434.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Britt RD Jr, Thompson MA, Wicher SA, Manlove LJ, Roesler A, Fang YH, Roos C, Smith L, Miller JD, Pabelick CM, Prakash YS. Smooth muscle brain-derived neurotrophic factor contributes to airway hyperreactivity in a mouse model of allergic asthma. FASEB J 33: 3024–3034, 2019. doi: 10.1096/fj.201801002R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Cao XH, Chen SR, Li L, Pan HL. Nerve injury increases brain-derived neurotrophic factor levels to suppress BK channel activity in primary sensory neurons. J Neurochem 121: 944–953, 2012. doi: 10.1111/j.1471-4159.2012.07736.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Chakder S, McHugh KM, Rattan S. Inhibitory neurotransmission in lethal spotted mutant mice: a model for Hirschsprung’s disease. Gastroenterology 112: 1575–1585, 1997. doi: 10.1016/S0016-5085(97)70039-8. [DOI] [PubMed] [Google Scholar]
  • 11.Chakder S, Rathi S, Ma XL, Rattan S. Heme oxygenase inhibitor zinc protoporphyrin IX causes an activation of nitric oxide synthase in the rabbit internal anal sphincter. J Pharmacol Exp Ther 277: 1376–1382, 1996. [PubMed] [Google Scholar]
  • 12.Chakder S, Rattan S. Release of nitric oxide by activation of nonadrenergic noncholinergic neurons of internal anal sphincter. Am J Physiol 264: G7–G12, 1993. doi: 10.1152/ajpgi.1993.264.1.G7. [DOI] [PubMed] [Google Scholar]
  • 13.Coulie B, Szarka LA, Camilleri M, Burton DD, McKinzie S, Stambler N, Cedarbaum JM. Recombinant human neurotrophic factors accelerate colonic transit and relieve constipation in humans. Gastroenterology 119: 41–50, 2000. doi: 10.1053/gast.2000.8553. [DOI] [PubMed] [Google Scholar]
  • 14.De Godoy MA, Dunn S, Rattan S. Evidence for the role of angiotensin II biosynthesis in the rat internal anal sphincter tone. Gastroenterology 127: 127–138, 2004. doi: 10.1053/j.gastro.2004.03.056. [DOI] [PubMed] [Google Scholar]
  • 15.de Godoy MA, Rattan N, Rattan S. Arachidonic acid metabolites follow the preferential course of cyclooxygenase pathway for the basal tone in the internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 296: G727–G734, 2009. doi: 10.1152/ajpgi.90707.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Endo Y, Shirai T, Akamatsu T, Asada K. Comparison of fractional exhaled nitric oxide levels measured using the NIOX VERO and NOA 280i. Ann Allergy Asthma Immunol 119: 383–385, 2017. doi: 10.1016/j.anai.2017.07.026. [DOI] [PubMed] [Google Scholar]
  • 17.Esteban I, Hannestad J, Levanti B, Del Valle ME, Naves FJ, Vega JA. Neurotrophin receptor proteins immunoreactivity in human gastrointestinal endocrine cells. Brain Res Bull 38: 539–543, 1995. doi: 10.1016/0361-9230(95)02025-9. [DOI] [PubMed] [Google Scholar]
  • 18.Fan Y-P, Puri RN, Rattan S. Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action. Am J Physiol Gastrointest Liver Physiol 282: G461–G469, 2002. doi: 10.1152/ajpgi.00207.2001. [DOI] [PubMed] [Google Scholar]
  • 19.Free RB, Clark J, Amara S, Sibley DR. Neurotransmission in the central nervous system. In: Goodman & Gilman’s: The Pharmacological Basis of Therapeutics, edited by Brunton LL, Hilal-Dandan R, Knollmann BC. New York: McGraw-Hill Education, 2018. [Google Scholar]
  • 20.Freeman MR, Sathish V, Manlove L, Wang S, Britt RD Jr, Thompson MA, Pabelick CM, Prakash YS. Brain-derived neurotrophic factor and airway fibrosis in asthma. Am J Physiol Lung Cell Mol Physiol 313: L360–L370, 2017. doi: 10.1152/ajplung.00580.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Fu Y, Lin YM, Winston JH, Radhakrishnan R, Huang LM, Shi XZ. Role of brain-derived neurotrophic factor in the pathogenesis of distention-associated abdominal pain in bowel obstruction. Neurogastroenterol Motil 30: e13373, 2018. doi: 10.1111/nmo.13373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Grider JR, Piland BE, Gulick MA, Qiao LY. Brain-derived neurotrophic factor augments peristalsis by augmenting 5-HT and calcitonin gene-related peptide release. Gastroenterology 130: 771–780, 2006. doi: 10.1053/j.gastro.2005.12.026. [DOI] [PubMed] [Google Scholar]
  • 23.Hempstead BL. Brain-derived neurotrophic factor: three ligands, many actions. Trans Am Clin Climatol Assoc 126: 9–19, 2015. [PMC free article] [PubMed] [Google Scholar]
  • 24.Heyt GJ, Oh MK, Alemzadeh N, Rivera S, Jimenez SA, Rattan S, Cohen S, Dimarino AJ Jr. Impaired rectoanal inhibitory response in scleroderma (systemic sclerosis): an association with fecal incontinence. Dig Dis Sci 49: 1040–1045, 2004. doi: 10.1023/B:DDAS.0000034569.85066.69. [DOI] [PubMed] [Google Scholar]
  • 25.Hoehner JC, Wester T, Påhlman S, Olsen L. Alterations in neurotrophin and neurotrophin-receptor localization in Hirschsprung’s disease. J Pediatr Surg 31: 1524–1529, 1996. doi: 10.1016/S0022-3468(96)90170-0. [DOI] [PubMed] [Google Scholar]
  • 26.Hoehner JC, Wester T, Påhlman S, Olsen L. Localization of neurotrophins and their high-affinity receptors during human enteric nervous system development. Gastroenterology 110: 756–767, 1996. doi: 10.1053/gast.1996.v110.pm8608885. [DOI] [PubMed] [Google Scholar]
  • 27.Holschneider AM, Puri P. Hirschsprung’s Disease and Allied Disorders. Berlin: Springer-Verlag, 2008, p. 1–414. [Google Scholar]
  • 28.Katsui R, Kojima Y, Kuniyasu H, Shimizu J, Koyama F, Fujii H, Nakajima Y, Takaki M. A new possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system. Am J Physiol Gastrointest Liver Physiol 294: G1084–G1093, 2008. doi: 10.1152/ajpgi.00345.2007. [DOI] [PubMed] [Google Scholar]
  • 29.Liu S. Neurotrophic factors in enteric physiology and pathophysiology. Neurogastroenterol Motil 30: e13446, 2018. doi: 10.1111/nmo.13446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lommatzsch M, Braun A, Mannsfeldt A, Botchkarev VA, Botchkareva NV, Paus R, Fischer A, Lewin GR, Renz H. Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived neurotrophic functions. Am J Pathol 155: 1183–1193, 1999. doi: 10.1016/S0002-9440(10)65221-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Millar AJ, Steinberg RM, Raad J, Rode H. Anal achalasia after pull-through operations for Hirschsprung’s disease–preliminary experience with topical nitric oxide. Eur J Pediatr Surg 12: 207–211, 2002. doi: 10.1055/s-2002-32722. [DOI] [PubMed] [Google Scholar]
  • 32.Mohanty I, Singh J, Rattan S. Downregulation of thromboxane A2 and angiotensin II type 1 receptors associated with aging-related decrease in internal anal sphincter tone. Sci Rep 9: 6759, 2019. doi: 10.1038/s41598-019-42894-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Patapoutian A, Reichardt LF. Trk receptors: mediators of neurotrophin action. Curr Opin Neurobiol 11: 272–280, 2001. doi: 10.1016/S0959-4388(00)00208-7. [DOI] [PubMed] [Google Scholar]
  • 34.Pezet S, McMahon SB. Neurotrophins: mediators and modulators of pain. Annu Rev Neurosci 29: 507–538, 2006. doi: 10.1146/annurev.neuro.29.051605.112929. [DOI] [PubMed] [Google Scholar]
  • 35.Prakash YS, Martin RJ. Brain-derived neurotrophic factor in the airways. Pharmacol Ther 143: 74–86, 2014. doi: 10.1016/j.pharmthera.2014.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Rao SS. Constipation: evaluation and treatment. Gastroenterol Clin North Am 32: 659–683, 2003. doi: 10.1016/S0889-8553(03)00026-8. [DOI] [PubMed] [Google Scholar]
  • 37.Rao SS. Pathophysiology of adult fecal incontinence. Gastroenterology 126, Suppl 1: S14–S22, 2004. doi: 10.1053/j.gastro.2003.10.013. [DOI] [PubMed] [Google Scholar]
  • 38.Rattan S. Ca2+/calmodulin/MLCK pathway initiates, and RhoA/ROCK maintains, the internal anal sphincter smooth muscle tone. Am J Physiol Gastrointest Liver Physiol 312: G63–G66, 2017. doi: 10.1152/ajpgi.00370.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Rattan S, Phillips BR, Maxwell PJ IV. RhoA/Rho-kinase: pathophysiologic and therapeutic implications in gastrointestinal smooth muscle tone and relaxation. Gastroenterology 138: 13–8.e3, 2010. doi: 10.1053/j.gastro.2009.11.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Rattan S, Chakder S. Role of nitric oxide as a mediator of internal anal sphincter relaxation. Am J Physiol 262: G107–G112, 1992. doi: 10.1152/ajpgi.1992.262.1.G107. [DOI] [PubMed] [Google Scholar]
  • 41.Rattan S, De Godoy MA, Patel CA. Rho kinase as a novel molecular therapeutic target for hypertensive internal anal sphincter. Gastroenterology 131: 108–116, 2006. doi: 10.1053/j.gastro.2006.03.043. [DOI] [PubMed] [Google Scholar]
  • 42.Rattan S, Regan RF, Patel CA, De Godoy MA. Nitric oxide not carbon monoxide mediates nonadrenergic noncholinergic relaxation in the murine internal anal sphincter. Gastroenterology 129: 1954–1966, 2005. doi: 10.1053/j.gastro.2005.08.050. [DOI] [PubMed] [Google Scholar]
  • 43.Rattan S, Sarkar A, Chakder S. Nitric oxide pathway in rectoanal inhibitory reflex of opossum internal anal sphincter. Gastroenterology 103: 43–50, 1992. doi: 10.1016/0016-5085(92)91093-J. [DOI] [PubMed] [Google Scholar]
  • 44.Rattan S, Singh J, Kumar S, Phillips B. Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans. Am J Physiol Gastrointest Liver Physiol 308: G924–G933, 2015. doi: 10.1152/ajpgi.00017.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Sanders KM, Ward SM. Nitric oxide and its role as a non-adrenergic, non-cholinergic inhibitory neurotransmitter in the gastrointestinal tract. Br J Pharmacol 176: 212–227, 2019. doi: 10.1111/bph.14459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Schriemer D, Sribudiani Y, IJpma A, Natarajan D, MacKenzie KC, Metzger M, Binder E, Burns AJ, Thapar N, Hofstra RMW, Eggen BJL. Regulators of gene expression in enteric neural crest cells are putative Hirschsprung disease genes. Dev Biol 416: 255–265, 2016. doi: 10.1016/j.ydbio.2016.06.004. [DOI] [PubMed] [Google Scholar]
  • 47.Singh J, Boopathi E, Addya S, Phillips B, Rigoutsos I, Penn RB, Rattan S. Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: role of microRNA-133a. Am J Physiol Gastrointest Liver Physiol 311: G964–G973, 2016. doi: 10.1152/ajpgi.00290.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Singh J, Cohen S, Mehendiratta V, Mendoza F, Jimenez SA, Dimarino AJ, Rattan S. Effects of scleroderma antibodies and pooled human immunoglobulin on anal sphincter and colonic smooth muscle function. Gastroenterology 143: 1308–1318, 2012. doi: 10.1053/j.gastro.2012.07.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Singh J, Mehendiratta V, Del Galdo F, Jimenez SA, Cohen S, DiMarino AJ, Rattan S. Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 297: G1206–G1213, 2009. doi: 10.1152/ajpgi.00286.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Singh J, Mohanty I, Addya S, Phillips B, Mee Yong H, An SS, Penn RB, Rattan S. Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone. Sci Rep 7: 1477–1487, 2017. doi: 10.1038/s41598-017-01550-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Singh J, Rattan S. Role of PKC and RhoA/ROCK pathways in the spontaneous phasic activity in the rectal smooth muscle. Am J Physiol Gastrointest Liver Physiol 304: G723–G731, 2013. doi: 10.1152/ajpgi.00473.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Skaper SD. The neurotrophin family of neurotrophic factors: an overview. Methods Mol Biol 846: 1–12, 2012. doi: 10.1007/978-1-61779-536-7_1. [DOI] [PubMed] [Google Scholar]
  • 53.Skaper SD. Neurotrophic factors: an overview. Methods Mol Biol 1727: 1–17, 2018. doi: 10.1007/978-1-4939-7571-6_1. [DOI] [PubMed] [Google Scholar]
  • 54.Terauchi A, Kobayashi D, Mashimo H. Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation. Am J Physiol Gastrointest Liver Physiol 289: G291–G299, 2005. doi: 10.1152/ajpgi.00005.2005. [DOI] [PubMed] [Google Scholar]
  • 55.Wu JF, Lu CH, Yang CH, Tsai IJ. Diagnostic role of anal sphincter relaxation integral in high-resolution anorectal manometry for Hirschsprung disease in infants. J Pediatr 194: 136–141.e2, 2018. doi: 10.1016/j.jpeds.2017.10.017. [DOI] [PubMed] [Google Scholar]
  • 56.Zhao J, Du J, Pan Y, Chen T, Zhao L, Zhu Y, Chen Y, Zheng Y, Liu Y, Sun L, Hang P, Du Z. Activation of cardiac TrkB receptor by its small molecular agonist 7,8-dihydroxyflavone inhibits doxorubicin-induced cardiotoxicity via enhancing mitochondrial oxidative phosphorylation. Free Radic Biol Med 130: 557–567, 2019. doi: 10.1016/j.freeradbiomed.2018.11.024. [DOI] [PubMed] [Google Scholar]

Articles from American Journal of Physiology - Gastrointestinal and Liver Physiology are provided here courtesy of American Physiological Society

RESOURCES