Activity of Protease-Activated Receptors in the Human Submucous Plexus

doi:10.1053/j.gastro.2011.08.034

Gastroenterology

Volume 141, Issue 6, December 2011, Pages 2088-2097.e1

https://doi.org/10.1053/j.gastro.2011.08.034 Get rights and content

Background & Aims

Protease-activated receptors (PARs) are expressed in the enteric nervous system. Excessive release of proteases has been reported in functional and inflammatory bowel diseases. Studies in several animal models indicate the involvement of neural PARs. We studied the actions of different PAR-activating peptides (AP) in the human submucous plexus and performed comparative studies in guinea pig submucous neurons.

Methods

We used voltage- and calcium-sensitive dye recordings to study the effects of PAR1-AP, PAR2-AP, PAR4-AP, the PAR1 activator thrombin, and the PAR2 activator tryptase on neurons and glia in human and guinea pig submucous plexus. Human preparations were derived from surgical resections. Levels of mucosal secretion evoked by PAR-APs were measured in Ussing chambers.

Results

PAR1-AP and thrombin evoked a prominent spike discharge and intracellular Ca²⁺ concentration ([Ca]_i) transients in most human submucous neurons and glia. PAR2-AP, tryptase, and PAR4-AP caused significantly weaker responses in a minor population. In contrast, PAR2-AP evoked much stronger responses in enteric neurons and glia of guinea pigs than did PAR1-AP or PAR4-AP. PAR1-AP, but not PAR2-AP or PAR4-AP, evoked a nerve-mediated secretion in human epithelium. The PAR1 antagonist SCH79797 inhibited the PAR1-AP, and thrombin evoked responses on neurons, glia, and epithelial secretion. In the submucous layer of human intestine, but not guinea pig intestine, PAR2-AP evoked [Ca]_i signals in CD68⁺ macrophages.

Conclusions

In the human submucous plexus, PAR1, rather than PAR2 or PAR4, activates nerves and glia. These findings indicate that PAR1 should be the focus of future studies on neural PAR-mediated actions in the human intestine; PAR1 might be developed as a therapeutic target for gastrointestinal disorders associated with increased levels of proteases.

Section snippets

Tissue Samples

Tissue samples were obtained from 58 male guinea pigs (Dunkin Hartley; Harlan Winkelmann, Borchen, Germany). For the neuroimaging studies, colonic segments were removed after killing the animals by cervical dislocation and exsanguinations in accordance with German ethical guidelines. Segments were dissected in ice-cold oxygenated (95% o₂ and 5% co₂, pH 7.4) Krebs solution containing (in mmol/L): 117 NaCl, 4.7 KCl, 1.2 MgCl₂.6H₂0, 1.2 NaH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂.2H₂0, and 11 glucose. The

PAR-APs Evoked Spike Discharge in Submucous Neurons

Microejection of PAR1-AP onto human submucous ganglia induced a median spike discharge of 2.1 Hz in 71.4% of enteric neurons (on average, 3 neurons per ganglion). The PAR1-AP activated a higher proportion of neurons with a significantly higher spike discharge compared with PAR4-AP (0.8 Hz; 33.3% responding neurons) or PAR2-AP (0.8 Hz; 6.3% responding neurons) (Figure 1). Even longer applications of PAR2-AP for 30 seconds did not increase the number of responding neurons (median, 0%) or the

Discussion

This is the first report describing the effects of PAR activation in the human enteric nervous system as well as in guinea pig submucous plexus. Our study revealed that stimulation of PARs by PAR selective peptides and by the endogenous proteases thrombin and tryptase activated neurons and glia in the human submucous plexus. The most striking result was that most neurons and glia in human submucous plexus responded to PAR1-AP and thrombin application, whereas activation of PAR2 and PAR4 yielded

References (36)

D.R. Linden et al.
Agonists of proteinase-activated receptor 2 excite guina pig ileal myenteric neurons
Eur J Pharmacol
(2001)
C. Gao et al.
Serine proteases excite myenteric neurons through protease-activated receptors in guinea pig small intestine
Gastroenterology
(2002)
N. Cenac et al.
Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2
Am J Pathol
(2002)
R. Roka et al.
A pilot study of fecal serine-protease activity: a pathophysiologic factor in diarrhea-predominant irritable bowel syndrome
Clin Gastroenterol Heptatol
(2007)
A. Annaházi et al.
Fecal proteases from diarrheic-IBS and ulcerative colitis patients exert opposite effect on visceral sensitivity in mice
Pain
(2009)
G. Barbara et al.
Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome
Gastroenterology
(2004)
S. Buhner et al.
Activation of human enteric neurons by supernatants of colonic specimens from patients with irritable bowel syndrome
Gastroenterology
(2009)
G. Barbara et al.
Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome
Gastroenterology
(2007)
K. Michel et al.
Serotonin excites neurons in the human submucous plexus via 5-HT3 receptors
Gastroenterology
(2005)
M. Schemann et al.
The beta3-adrenoreceptor agonist GW427353 (Solabegron) decreases excitability of human enteric neurons via release of somatostatin
Gastroenterology
(2010)

H.S. Ahn et al.

Inhibition of cellular action of thrombin by N3-Cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]quinazoline-1,3-diamine (SCH79797), a nonpeptide thrombin receptor antagonist

Biochem Pharmacol

(2000)

S. Saibeni et al.

Increased thrombin generation in inflammatory bowel diseases

Thromb Res

(2010)

N. Vergnolle

Clinical relevance of proteinase activated receptors (pars) in the gut

Gut

(2005)

M.D. Hollenberg

Getting the message across: pathophysiology and signalling via receptors for polypeptide hormones and proteinases

Clin Invest Med

(2010)

M.D. Hollenberg et al.

International Union of PharmacologyXXVIII. Proteinase-activated receptors

Pharmacol Rev

(2002)

D.E. Reed et al.

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

J Physiol

(2003)

C.U. Covera et al.

Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and −2

J Physiol

(1999)

R. Garrido et al.

Presence of functionally active protease-activated receptors 1 and 2 in myenteric glia

J Neurochem

(2002)

Cited by (40)

The Eph/ephrin system symphony of gut inflammation
2023, Pharmacological Research
The extent of gut inflammation depends largely on the gut barrier’s integrity and enteric neuroimmune interactions. However, the factors and molecular mechanisms that regulate inflammation-related changes in the enteric nervous system (ENS) remain largely unexplored. Eph/ephrin signaling is critical for inflammatory response, neuronal activation, and synaptic plasticity in the brain, but its presence and function in the ENS have been largely unknown to date. This review discusses the critical role of Eph/ephrin in regulating gut homeostasis, inflammation, neuroimmune interactions, and pain pathways. Targeting the Eph/ephrin system offers innovative treatments for gut inflammation disorders, offering hope for enhanced patient prognosis, pain management, and overall quality of life.
Proteinase-activated receptors regulate intestinal functions in a segment-dependent manner in rats
2022, European Journal of Pharmacology
Citation Excerpt :
Indeed, immunocytochemical stainings revealed the expression of both PAR1 and PAR2 in ganglionic cells which were immunoreactive for the neuronal marker PGP9.5 as well as for PAR1 and PAR2. Functionally, stimulation of PAR1 and PAR2 is concomitant with an increase in the cytosolic Ca2+ concentration in guinea-pig submucous neurons, whereas in human submucous neurons, this is only the case for PAR1 (Mueller et al., 2001). A similar phenomenon was observed with rat myenteric neurons investigated in the present study: only stimulation of PAR1 with thrombin (Fig. 8), but not of PAR2 with trypsin resulted in an increase in the cytosolic Ca2+ concentration.
Proteinases released e.g. during inflammatory or allergic responses affect gastrointestinal functions via proteinase-activated receptors such as PAR1 and PAR2. As the gastrointestinal tract exerts pronounced gradients along its longitudinal axis, the present study focuses on the effect of PAR1 and PAR2 agonists on electrogenic ion transport (measured as short-circuit current; I_sc), tissue conductance (G_t) and contractility of the longitudinal muscle layer of rats. In Ussing chamber experiments, the PAR1 agonist TFLLR-NH₂, which mimics the tethered ligand liberated after cleavage of the receptor, evoked only a modest increase in I_sc (<0.5 μEq·h⁻¹·cm⁻²) in small intestine, but a strong increase (3–4 μEq·h⁻¹·cm⁻²) in colon. Pretreatment with tetrodotoxin reduced the response of the colonic segments to the level of the small intestine. Thrombin, the natural activator of PAR1, was much less effective suggesting biased activation by this peptidase. A similar gradient along the longitudinal axis of the intestine was observed with trypsin, the endogenous activator of PAR2. Divergent actions of PAR1 activation by enzymatic cleavage or a mimetic peptide were also observed when recording isometric contractions of longitudinal muscle. For example, in the jejunum TFLLR-NH₂ concentration-dependently induced a contractile response, whereas thrombin showed only inconsistent effects. The PAR2 activator AC264613 induced a concentration-dependent decrease in muscle tone combined with an inhibition of phasic spontaneous contractions. PCR experiments and immunohistochemical stainings confirmed the expression of PAR1 and PAR2. The data implies that PAR1 and PAR2 functions vary depending on the intestinal segment.
Reprint of: Role of enteric neurotransmission in host defense and protection of the gastrointestinal tract
2014, Autonomic Neuroscience: Basic and Clinical
Citation Excerpt :
They are activated by proteolytic cleavage of the N-terminus, which allows the ligand domain to bind to the receptor. PAR1, PAR2 and PAR4 activate enteric neurons, enteric glia and extrinsic primary afferents, and the responses of that occur following this activation form part of the host defense response (Green et al., 2000; Buresi et al., 2005; Mueller et al., 2011; Buhner et al., 2012; Kugler et al., 2012). Notable, as it pertains to disease, is that supernatants of biopsies taken from patients with irritable bowel syndrome (IBS) and IBD have elevated levels of protease activity which is thought to contribute to disease pathogenesis and symptom generation in these conditions (Buhner et al., 2012).
Host defense is a vital role played by the gastrointestinal tract. As host to an enormous and diverse microbiome, the gut has evolved an elaborate array of chemical and physicals barriers that allow the digestion and absorption of nutrients without compromising the mammalian host. The control of such barrier functions requires the integration of neural, humoral, paracrine and immune signaling, involving redundant and overlapping mechanisms to ensure, under most circumstances, the integrity of the gastrointestinal epithelial barrier. Here we focus on selected recent developments in the autonomic neural control of host defense functions used in the protection of the gut from luminal agents, and discuss how the microbiota may potentially play a role in enteric neurotransmission. Key recent findings include: the important role played by subepithelial enteric glia in modulating intestinal barrier function, identification of stress-induced mechanisms evoking barrier breakdown, neural regulation of epithelial cell proliferation, the role of afferent and efferent vagal pathways in regulating barrier function, direct evidence for bacterial communication to the enteric nervous system, and microbial sources of enteric neurotransmitters. We discuss these new and interesting developments in our understanding of the role of the autonomic nervous system in gastrointestinal host defense.
Role of enteric neurotransmission in host defense and protection of the gastrointestinal tract
2014, Autonomic Neuroscience: Basic and Clinical
Citation Excerpt :
They are activated by proteolytic cleavage of the N-terminus, which allows the ligand domain to bind to the receptor. PAR1, PAR2 and PAR4 activate enteric neurons, enteric glia and extrinsic primary afferents, and the responses of that occur following this activation form part of the host defense response (Green et al., 2000; Buresi et al., 2005; Mueller et al., 2011; Buhner et al., 2012; Kugler et al., 2012). Notable, as it pertains to disease, is that supernatants of biopsies taken from patients with irritable bowel syndrome (IBS) and IBD have elevated levels of protease activity which is thought to contribute to disease pathogenesis and symptom generation in these conditions (Buhner et al., 2012).
Host defense is a vital role played by the gastrointestinal tract. As host to an enormous and diverse microbiome, the gut has evolved an elaborate array of chemical and physicals barriers that allow the digestion and absorption of nutrients without compromising the mammalian host. The control of such barrier functions requires the integration of neural, humoral, paracrine and immune signaling, involving redundant and overlapping mechanisms to ensure, under most circumstances, the integrity of the gastrointestinal epithelial barrier. Here we focus on selected recent developments in the autonomic neural control of host defense functions used in the protection of the gut from luminal agents, and discuss how the microbiota may potentially play a role in enteric neurotransmission. Key recent findings include: the important role played by subepithelial enteric glia in modulating intestinal barrier function, identification of stress-induced mechanisms evoking barrier breakdown, neural regulation of epithelial cell proliferation, the role of afferent and efferent vagal pathways in regulating barrier function, direct evidence for bacterial communication to the enteric nervous system, and microbial sources of enteric neurotransmitters. We discuss these new and interesting developments in our understanding of the role of the autonomic nervous system in gastrointestinal host defense.
The enteric nervous system
2023, Physiological Reviews
Inhibition of Serine Proteases as a Novel Therapeutic Strategy for Abdominal Pain in IBS
2022, Frontiers in Physiology

View all citing articles on Scopus

: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by the EU FP7-IPODD Consortium and the Deutsche Forschungsgemeinschaft (Sche-267/7-2 and GRK1482).

View full text

Original ResearchBasic and Translational—Alimentary TractActivity of Protease-Activated Receptors in the Human Submucous Plexus

Background & Aims

Methods

Results

Conclusions

Section snippets

Tissue Samples

PAR-APs Evoked Spike Discharge in Submucous Neurons

Discussion

Eur J Pharmacol

Gastroenterology

Am J Pathol

Clin Gastroenterol Heptatol

Pain

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Biochem Pharmacol

Thromb Res

Clinical relevance of proteinase activated receptors (pars) in the gut

Gut

Getting the message across: pathophysiology and signalling via receptors for polypeptide hormones and proteinases

Clin Invest Med

International Union of PharmacologyXXVIII. Proteinase-activated receptors

Pharmacol Rev

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

J Physiol

Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and −2

J Physiol

Presence of functionally active protease-activated receptors 1 and 2 in myenteric glia

J Neurochem

Original Research
Basic and Translational—Alimentary Tract
Activity of Protease-Activated Receptors in the Human Submucous Plexus