Rho Kinase as a Novel Molecular Therapeutic Target for Hypertensive Internal Anal Sphincter

doi:10.1053/j.gastro.2006.03.043

Gastroenterology

Volume 131, Issue 1, July 2006, Pages 108-116

https://doi.org/10.1053/j.gastro.2006.03.043 Get rights and content

Background & Aims: An increase in Rho kinase (ROK) activity has been associated with agonist-induced sustained contraction of the smooth muscle, but its role in the pathophysiology of spontaneously tonic smooth muscle is not known. Methods: Present studies examined the effects of ROK inhibitor Y-27632 in the tonic smooth muscle of the rat internal anal sphincter (IAS) versus in the flanking phasic smooth muscle of the rectum. In addition, studies were performed to determine the relationship between the decreases in the basal IAS tone and the ROK activity. Confocal microscopic studies determined the cellular distribution of the smooth muscle–predominant isoform of ROK (ROCK-II) in the smooth muscle cells (SMCs). Results: In in vitro studies using neurohumoral inhibitors and tetrodotoxin and the use of SMCs demonstrate direct relaxation of the IAS SMCs by Y-27632. The ROK inhibitor was more potent in the IAS than in the rectal smooth muscle. The IAS relaxation by Y-27632 correlated specifically with the decrease in ROK activity. Confocal microscopy revealed high levels of ROCK-II toward the periphery of the IAS SMCs. In in vivo studies, the lower doses of Y-27632 caused a potent and selective decrease in the IAS pressures without any adverse cardiovascular systemic effects. The ROK inhibitor also caused potent relaxation of the hypertensive IAS. Conclusions: RhoA/ROK play a crucial role in the maintenance of the basal tone in the IAS, and ROK inhibitors have a therapeutic potential in the IAS dysfunction characterized by the hypertensive IAS.

Section snippets

Measurement of Isometric Tension

Sprague–Dawley rats (300–350 g) were killed by decapitation, and the anal canal with an adjacent region of the rectum was quickly removed and transferred to oxygenated (95% o₂/5% co₂) Krebs physiologic solution of the following composition (in mmol/L): 118.07 NaCl, 4.69 KCl, 2.52 CaCl₂, 1.16 MgSO₄, 1.01 NaH₂PO₄, 25 NaHCO₃, and 11.10 glucose (37°C). Circular smooth muscle strips (∼0.5 × 7 mm) of the IAS and the rectal smooth muscle (RSM) were prepared as explained previously²⁸ and transferred to

Effect of Y-27632 on the Basal Tone of the IAS Versus the Phasic Activity of the RSM

The ROK inhibitor caused a concentration-dependent decrease in the basal tone of the IAS (Figure 1) with ∼30-fold higher potency than in the phasic smooth muscle of the RSM. The median effective concentrations in causing the decrease in the IAS tone versus in the phasic activity of the RSM were 0.44 μmol/L and 13 μmol/L, respectively. Y-27632 (1 μmol/L) caused 50% inhibition in the basal IAS tone without a significant effect in the RSM. Typical tracings of the effects of Y-27632 in the IAS and

Discussion

The studies show that the basal tone in the IAS is critically dependent on RhoA/ROK. The selectivity and potency of the ROK inhibition in causing IAS relaxation without significant side effects and its effectiveness in the hypertensive IAS suggest a therapeutic potential of this approach in certain conditions associated with the hypertensive IAS.

The relaxant effect of ROK inhibition works directly at the SMCs due to the lack of effect of different neurohumoral inhibitors and the neurotoxin

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To summarise current knowledge of Internal anal sphincter.
The internal anal sphincter (IAS) is the involuntary ring of smooth muscle in the anal canal and is the major contributor to the resting pressure in the anus. Structural injury or functional weakness of the muscle results in passive incontinence of faeces and flatus. With advent of new assessment and treatment modalities IAS has become an important topic for surgeons. This review was undertaken to summarise our current knowledge of internal anal sphincter and highlight the areas that need further research.
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C-type atriuretic peptide causes relaxation of the internal anal sphincter through natriuretic peptide receptor B
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Normal rabbit immunoglobulin G (IgG) was purchased from Dako (Copenhagen, Denmark). Measurements of relaxation of muscle strips from the IAS were performed according to a procedure published previously with minor modifications [20–22]. Male rats, weighing 300–350 g, were euthanized with CO2.
The internal anal sphincter (IAS) plays an important role in maintaining continence. Atrial natriuretic peptide (ANP) could relax the IAS. The purpose of the present study was to investigate the roles of natriuretic peptide receptors (NPRs) in the IAS.
Relaxation of isolated rat IAS strips caused by natriuretic peptides was measured using isometric transducers. Expression of NPRs was evaluated by reverse transcription–polymerase chain reaction (PCR), immunohistochemistry and real-time PCR.
In the rat IAS, C-type natriuretic peptide (CNP) produced a marked and concentration-dependent relaxation while ANP caused a mild relaxation. By contrast, brain natriuretic peptide, dendroaspis natriuretic peptide, and des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4–23) amide did not generate relaxation. CNP was much more effective than ANP and brain natriuretic peptide in stimulating the relaxation, indicating that NPR-B mediates IAS relaxation. The CNP-induced relaxation was inhibited by the protein kinase G inhibitor Rp-8CPT-cGMPS and potassium channel blocker charybdotoxin but not by protein kinase A inhibitor Rp-cAMPS. This suggests the involvement of cGMP and calcium-activated potassium channels in the CNP-induced IAS relaxation. Reverse transcription PCR and immunohistochemistry revealed the existence of NPR-B in the rat IAS. Furthermore, real-time PCR identified abundant expression of CNP and NPR-B in the rat IAS.
CNP causes relaxation of the IAS via NPR-B, cGMP, and potassium channel pathways in rats. CNP might regulate IAS motility. NPR-B is a potential therapeutic target in anorectal motility disorders.
Effects of scleroderma antibodies and pooled human immunoglobulin on anal sphincter and colonic smooth muscle function
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The composition of Krebs physiological solution (in mmol/L) was as follows: 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. The colonic smooth muscle strips (∼0.5 × 7 mm) from the circular smooth muscle layer were prepared for the recording of isometric tension using 2-mL organ baths as described previously.24,25 These smooth muscle strips then were transferred to 2-mL organ baths containing oxygenated Krebs physiological solution and were allowed to equilibrate for 1–2 hours.
Patients with systemic sclerosis (SSc) have impairments in gastrointestinal smooth muscle function. The disorder has been associated with circulating antibodies to cholinergic muscarinic the type-3 receptor (M₃-R). We investigated whether it is possible to neutralize these antibodies with pooled human IgGs (pooledhIgG).
We studied the effects of IgGs purified from patients with SSc (SScIgGs) on cholinergic nerve stimulation in rat colon tissues. We also examined the effects of SScIgGs on M₃-R activation by bethanechol (BeCh), M₃-R occupancy, and receptor binding using immunofluorescence, immunoblot, and enzyme-linked immunosorbent analyses of human internal anal sphincter (IAS) smooth muscle cells, before and after administration of pooledhIgG. Functional displacement of M₃-R occupancy by the SScIgGs was compared with that of other IgGs during the sustained phase of BeCh-induced contraction of intact smooth muscles from rats.
SScIgG significantly attenuated neurally mediated contraction and acetylcholine release in rat colon as well as BeCh-induced sustained contraction of the IAS smooth muscle. In immunofluorescence analysis, SScIgG co-localized with M₃-R. In immunoblot and enzyme-linked immunosorbent analyses, M₃-R loop-2 peptide and human IAS SMC membrane lysates bound significant amounts of SScIgG, compared with IgGs from healthy individuals and pooledhIgG. Binding was attenuated significantly by application of pooledhIgG, which by itself had no significant effect. Incubation of samples with pooledhIgG, or mixing pooledhIgG with SScIgG before administration to tissues, significantly reduced binding of SScIgG, indicating that pooledhIgG prevents SScIgG blockade of M₃-R.
In studies of rat and human tissues, pooled human IgG prevent and reverses the cholinergic dysfunction associated with the progressive gastrointestinal manifestations of SSc by neutralizing functional M₃-R antibodies present in the circulation of patients with SSc.
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Earlier studies testing the effects of selective inhibition of PKC in the LES and IAS suggested PKC to be the primary molecular mechanism in the basal tone of the gastrointestinal smooth muscles [2,57,58]. Recent studies, however, aimed at assessing the relative contribution of ROCK vs PKC show that ROCK-mediated inhibition of MLCP is primarily responsible for the basal tone in the LES of humans and animals, and for the IAS of various animals [2,11,13,59,60]. The role of these pathways, however, in the intact human IAS remains to be determined.
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: Supported by National Institutes of Diabetes and Digestive and Kidney Diseases grant DK-35385 and an institutional grant from Thomas Jefferson University.

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Basic–alimentary tractRho Kinase as a Novel Molecular Therapeutic Target for Hypertensive Internal Anal Sphincter

Section snippets

Measurement of Isometric Tension

Effect of Y-27632 on the Basal Tone of the IAS Versus the Phasic Activity of the RSM

Discussion

J Pediatr Surg

Gastroenterology

Gastroenterology

Baillieres Best Pract Res Clin Gastroenterol

Gastroenterology

Gastroenterology

Gastroenterology

J Biol Chem

Trends Pharmacol Sci

Gastroenterology

Gastroenterology

Fecal incontinence

Constipation

The internal anal sphincterregulation of smooth muscle tone and relaxation

Neurogastroenterol Motil

Progress in the understanding and treatment of chronic anal fissure

Postgrad Med J

Genesis of anal canal pressures in the opossum

Am J Physiol Gastrointest Liver Physiol

Calcium-dependent and calcium-independent contractions in smooth muscles

Am J Med

Signal-transduction pathways that regulate smooth muscle function. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles

Am J Physiol Gastrointest Liver Physiol

MAPK mediates PKC-dependent contractions of cat esophageal and lower esophageal sphincter circular smooth muscle

Am J Physiol Gastrointest Liver Physiol

Biochem J

Sustained muscle contraction induced by agonists, growth factors, and Ca2+ mediated by distinct PKC isozymes

Am J Physiol Gastrointest Liver Physiol

Topical diltiazem ointment in the treatment of chronic anal fissure

Br J Surg

Basic–alimentary tract
Rho Kinase as a Novel Molecular Therapeutic Target for Hypertensive Internal Anal Sphincter

Sustained muscle contraction induced by agonists, growth factors, and Ca²⁺ mediated by distinct PKC isozymes