The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts

Abstract

1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.

Figure 1

Reproducibility and concentration dependency of calcium signal initiation at the base of human colonic crypts. (a) Phase microscopy of isolated human colonic crypts bounded by a monolayer of non-contaminated epithelial cells that surround the distinctive crypt lumen (left hand panel: objective. ×20; scale bar=30 μm. Right hand panel: objective ×40; scale bar=30 μm). A typical region of interest within which changes in intracellular calcium were monitored with respect to time is indicated by the open black box placed at the crypt-base in the right hand panel. (b) Calcium responses to successive pulses of stimulation by ACh (10 μM) are reproducible. The individual responses are shown to be superimposable in (c), see text for statistical details. (d) Illustration of the concentration dependency of initial rate, amplitude and latency of the ACh-induced calcium response. At the end of each crypt experiment a maximal response was invoked (achieved with 1 mM ACh), and the preceding responses normalised according to the corresponding parameter. Data from individual crypts are shown collated in the form of a concentration–normalized rate of response curve (e) (mean±s.e., n>3, absence of error bars indicates that magnitude of s.e. is less than the size of the data symbol). The data were best-fitted by a curve of first order kinetics and an EC₅₀ value of 14 μM.

Figure 2

ACh-induced colonic crypt calcium signals are initiated by mobilization of calcium from intracellular stores. (a) The rate and amplitude of calcium peak formation were superimposable in the presence or absence of calcium in the bathing medium (see text for statistical details). (b) Chronic (>1000 s exposure) thapsigargin (300 nM)-induced depletion of intracellular calcium stores ablated the response to the second exposure to ACh (10 μM). (c) Acute exposure to a lower concentration of thapsigargin (100 nM) generated a moderate increase in the intracellular calcium level, which did not inhibit the ACh response. (d) Successive ACh (10 μM)-induced calcium responses conducted in the absence (bold trace) or presence (light trace) of the calcium channel blocker nifedipine (1 μM, 15 min pre-incubation). The responses are of similar rate and amplitude (see text for statistical details).

Figure 3

Functional and immunohistochemical characterization of calcium-coupled muscarinic receptor subtypes in human colonic crypts. (a) Inhibition of ACh (10 μM) induced calcium response by the muscarinic receptor antagonist atropine (100 nM). (b) mAChR subtype selective antagonists inhibited the rate of calcium signal initiation in a dose dependent manner 4-DAMP <AF-DX 384 <pirenzepine <methroctamine. Normalized rate data were best fitted by inhibition curves (see equation 2, Methods) with IC₅₀ values (nM) of 0.78, 69, 128, 2510, respectively. Data points represent the mean±s.e.mean (n>3). (c) Immunohistochemical labelling of M₁, M₃ and M₅ muscarinic receptor subtypes. M₃ receptor labelling was evident along the basolateral membranes of cells located at the crypt base. See text for background corrected fluorescence intensities associated with the labelling of each receptor subtype.

Figure 4

Otilonium bromide inhibits acetylcholine-induced colonic crypt calcium signals in a dose dependent manner. (a) A real-time example of the control and experimental acetylcholine-induced calcium responses conducted in the absence and presence of OB, respectively. (b) Progressive inhibition of ACh (10 μM)-induced calcium responses at increasing concentrations of OB. Traces for the paired control and OB pre-treatment (10 nM, 1 μM, 100 μM) are shown superimposed. (c) For each concentration of OB the normalized rate of calcium increase, with respect to the paired control response, is plotted in the form of an inhibition curve. The data were fitted by equation 2 (IC₅₀=880 nM). Data points represent the mean±s.e.mean (n>3) and the illustrated traces are representative of at least four independent experiments.

Figure 5

Otilonium bromide inhibits recombinant M₃AChR coupled calcium signals, but not those induced by stimulation of endogenous purinergic and histaminic receptors. (a) Otilonium bromide (10 μM) ameliorated ACh (10 μM)-induced calcium signals in CHO-M₃ cells (i) but not those induced by activation of endogenous P₂Y₂ receptors (ii). (b) A bar chart of the initial rate of cytosolic calcium increase induced by each agonist in the presence of OB, normalized to the unpaired control rate in each case. (c) Histamine-induced human colonic crypt calcium responses were not inhibited by OB (10 μM). (d) Mean agonist (ACh and histamine both 10 μM)-induced rate of calcium increase in the presence of OB (10 μM) normalized to the paired control (*P<0.05, unpaired t-test, n⩾3 in each case).