Pharmacological evidence for putative CCK(1) receptor heterogeneity in human colon smooth muscle

Abstract

1. The pharmacology of the cholecystokinin CCK(1) receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. 2. Saturation analysis of the interaction between the radiolabelled, selective CCK(1)-receptor antagonist, [(3)H]-L-364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. 3. Competition studies, using a range of structurally diverse, CCK-receptor selective ligands provided further evidence for CCK(1) receptor heterogeneity in human colon tissue (n(H) values significantly less than unity for SR27897=0.77+/-0.07, 2-NAP=0.73+/-0.03, YM220=0.70+/-0.09 and PD-134,308=0.83+/-0.01). Moreover, the competition data for SR27897, 2-NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. 4. The data obtained are consistent with the presence of a single CCK(1) receptor binding site in the gallbladder but not in the colon. A two-site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established.

Figure 1

Total, non-specific and specific binding of [³H]-L-364,718 (0.1 nM; ∼8500 d.p.m. added plotted as a function of increasing gallbladder (a and b) and ascending colon (c and d) membrane concentration. Increasing concentrations (gallbladder; 0–180 mg ml⁻¹, ascending colon 0–120 mg ml⁻¹) of enriched membranes (400 □mu;l) were incubated, in triplicate, with 0.1 nM [³H]-L-364,718 (50 μl; 1 nM) for 150 min at 21±3°C. Total binding and non-specific binding were defined with 50 μl buffer B and 50 μl of 10 μM SR27897, respectively. Data represents the mean±s.e. mean of four experiments. The linear relationship between gallbladder membrane concentration (b) or ascending colon membrane concentration (d) and the specific binding of [³H]-L-364,718 is also shown (ii; hatched lines demonstrate the 95% confidence intervals of the linear regression).

Figure 2

Saturation analysis of the binding of [³H]-L-364,718 to sites in human gallbladder (a) and ascending colon (c) membranes. Tissue (400 μl; 20 mg ml⁻¹ gallbladder and 75 mg ml⁻¹ colon) was incubated in triplicate with increasing concentrations of [³H]-L-364,718 (50 μμl; 0.01–0.5 nM) and 50 μl of buffer B or 50 μμl of 10 μM SR27897 to define total and non-specific binding, respectively. The incubation was terminated after 150 min at 21±3°C. The transformed specific binding data is also shown in the corresponding Scatchard plots (b and d). Data are representative of four experiments.

Figure 3

Graphical representation of the binding kinetics of [³H]-L-364,718 in the human gallbladder (a) and ascending colon (b) assays (results from a single experiment are shown here). The association rate was measured at 21±3°C by incubating [³H]-L-364,718 (50 μl; 1 nM) for increasing times (0.5–60 min) with gallbladder (400 μl; 20 mg ml⁻¹) or ascending colon (400 μl; 20 mg ml⁻¹) membranes (in triplicate) and 50 μl of buffer B or 1 μM SR27897 to define the specific and non-specific binding, respectively. The dissociation rate for [³H]-L-364,718 in the CCK₁ receptor assay was determined by incubating [³H]-L-364,718 (50 μl; 1 nM) in sextruplicate with 50 μl of buffer B (total binding) and in triplicate with 50 μl of 1 μM SR27897 (non-specific binding) for 150 min at 21±3°C. At this time 10 μl of 50 μM SR27897 was added to a triplicate group of tubes (defining total binding) and the bound radioactivity was determined at increasing time intervals (0.5–100 min).

Figure 4

Competition between [³H]-L-364,718 (0.1 nM) and increasing concentrations of SR27897 (a), 2-NAP (b) and YM220 (c) in both the human gallbladder and the human ascending colon assays. Data represent the mean±s.e.mean of four experiments. The curves superimposed on the mean experimental data are the one-site model fit for the gallbladder data (solid line) and the two-site model fit for the ascending colon (hatched line).

Figure 5

Comparison of affinity estimates (pK_I values) obtained in the human gallbladder or human ascending colon binding assay. The line shown superimposed is the line of identity. The pK_I values between the gallbladder and colon were frame-shifted from the line of identity (see panel a, principal components analysis: y=x+c, F_1,24=0.098; y=x, F_1,24=14.6). Comparison of the pK_I (2) values for SR27897, YM220 and 2-NAP from the two-site model fit with the gallbladder pK_I values (b) revealed a significantly better correlation than that obtained for the estimated pK_I (1) values with the gallbladder affinity data (c; F_2,12=5.81). See text for details of the two-site analysis.