Interactions between cholecystokinin and opioids in the isolated guinea-pig ileum

Abstract

1. Although cholecystokinin octapeptide sulphate (CCK-8) activates the opioid system of isolated guinea-pig ileum (GPI) whether it activates the mu- or kappa-system, or both, remains unclear. Neither is it known whether CCK-8 influences the withdrawal responses in GPI preparations briefly exposed to opioid agonists. This study was designed to clarify whether CCK-8 activates mu- or kappa-opioid systems or both; and to investigate its effect on the withdrawal contractures in GPI exposed to mu- or kappa-agonists and on the development of tolerance to the withdrawal response. 2. In GPI exposed to CCK-8, the selective kappa-antagonist nor-binaltorphimine elicited contractile responses that were concentration-related to CCK-8 whereas the selective mu-antagonist cyprodime did not. 3. In GPI preparations briefly exposed to the selective mu-agonist, dermorphin, or the selective kappa-agonist, U-50, 488H, and then challenged with naloxone, CCK-8 strongly enhanced the withdrawal contractures. 4. During repeated opioid agonist/CCK-8/opioid antagonist tests tolerance to opioid-induced withdrawal responses did not develop. 5. These results show that CCK-8 preferentially activates the GPI kappa-opioid system and antagonizes the mechanism(s) that control the expression of acute dependence in the GPI.

Figure 1

Contractile responses to opioid antagonists in isolated GPI preparations pre-contracted with CCK-8. Tissue preparations were exposed to CCK-8 (2×10⁻⁹ M). Naloxone (N; 5.4×10⁻⁷ M), the selective μ-opioid antagonist, cyprodime (CYP; 1.4×10⁻⁶ M) and the selective κ-antagonist, nor-binaltorphimine (BNI; 3.4×10⁻⁸ M) were added at the declining tonic response to CCK-8. The tracings were recorded from three tissue preparations obtained from the same animal.

Figure 2

Effect of the selective CCK_A antagonist, PD-140,548, on the tonic response to CCK-8 and on the subsequent response to naloxone of isolated GPI preparations. Tissue preparations were first tested with CCK-8 (4×10⁻¹⁰ M). In subsequent tests the CCK_A antagonist, PD-140,548 (1.2–80×10⁻⁸ M) was added 5 min before CCK-8. Naloxone (5.4×10⁻⁷ M) was added 2 min after CCK-8. Contractures are expressed as percentages of maximum response to ACh. The values shown are means±s.e.mean. For each PD-140,548 concentration, 6–10 tissue preparations from three animals were used (for controls, 24 tissue preparations from six animals). ^**P<0.01 vs control tonic response to CCK-8; ^##P<0.01 vs control response to naloxone.

Figure 3

Isolated GPI responses to CCK-8 (tonic contracture) and cyprodime in repeated dermorphin/CCK-8/cyprodime tests. Each tissue preparation was first tested with CCK-8 (2×10⁻⁹ M) and cyprodime (1.4×10⁻⁶ M). Thereafter, five tests with dermorphin/CCK-8/cyprodime (dermorphin: 1.2×10⁻⁸ M; CCK-8: 2×10⁻⁹ M; cyprodime: 1.4×10⁻⁶ M) were performed. After each test, tissue preparations were washed three times and allowed to rest for 30 min before next test. Contractures are expressed as percentages of the maximum response to ACh. The values shown are the means (±s.e.mean) from nine tissue preparations coming from five animals. ^**P<0.01 vs Test I (one-way ANOVA for repeated measures, followed by Student-Newman-Keuls test).

Figure 4

Concentration-response curves of dermorphin (a) and U-50, 488H (b) for inhibition of the tonic response to CCK-8 and the increase of the response to naloxone in GPI preparations. Tissue preparations were first tested with CCK-8 (2×10⁻⁹ M) and naloxone (5.4×10⁻⁷ M), added 2 min after CCK-8. In subsequent tests, opioid agonists were added 2 min before CCK-8 (2×10⁻⁹ M); naloxone (5.4×10⁻⁷ M) was added 2 min after CCK-8. Contractures are expressed as percentages of the maximum response to ACh. The values shown are the means (±s.e.mean) of 6–12 experiments (n=42 for controls). For the dermorphin concentration-response curve, 24 tissue preparations coming from six animals were used (one or two dermorphin concentrations were tested on each preparation). For the U-50,488H curve, 18 preparations coming from six animals were used (two or three U-50,488H concentrations were tested on each preparation). ^**P<0.01 vs control tonic response to CCK-8; ^##P<0.01 vs response to naloxone in the CCK-8/naloxone tests.

Figure 5

Dermorphin (D, 3×10⁻⁹ M), added to the bath 2 min before CCK-8 (2×10⁻⁹ M), increases the response to naloxone (N; 5.4×10⁻⁷ M) of isolated guinea-pig ilea pre-contracted with CCK-8. The response to naloxone had the same intensity regardless of whether the antagonist was added at the declining tonic response to CCK-8 or 8 min after CCK-8, i.e. when the response to CCK-8 had faded. The tracings were obtained from the same tissue preparation.

Figure 6

A typical tracing showing that CCK-8 (4×10⁻¹⁰ M) strongly enhances the withdrawal response to naloxone (N; 5.4×10⁻⁷ M) after exposure to dermorphin (D; 1.2×10⁻⁹ M). The tracings were obtained from the same tissue preparation. After a first test with CCK-8/naloxone (CCK-8: 4×10⁻¹⁰ M; naloxone: 5.4×10⁻⁷ M), the preparation was exposed to dermorphin (1.2×10⁻⁹ M) for 5 min and then challenged with naloxone (5.4×10⁻⁷ M). In the subsequent test, the tissue preparation was exposed to dermorphin (1.2×10⁻⁹ M) and CCK-8, added 2 min after dermorphin; naloxone (5.4×10⁻⁷ M) was added at the declining tonic contraction to CCK-8.

Figure 7

Responses to naloxone following exposure to dermorphin in absence or presence of CCK-8 (2×10⁻⁹ M). The preparations were first tested with CCK-8/naloxone (CCK-8: 2×10⁻⁹ M; naloxone: 5.4×10⁻⁷ M). Tissue preparations were then exposed to dermorphin (1,2, 3 or 6×10⁻⁹ M) for 5 min and then challenged with naloxone (5.4×10⁻⁷ M). In the subsequent test, tissue preparations were exposed to dermorphin (same concentration as in the first test) and CCK-8, added 2 min after dermorphin; naloxone (5.4×10⁻⁷ M) was added at the declining tonic contraction to CCK-8. Contractures are expressed as percentages of ACh maximum. Values shown are means±s.e.mean. The number of tissue preparations tested for each dermorphin concentration is shown in brackets. Tissue preparations came from six animals. ^**P<0.01 vs the response to naloxone in dermorphin/naloxone tests; ^##P<0.01 vs the response to naloxone in the CCK-8/naloxone tests (one-way ANOVA for repeated measures, followed by Student-Newman-Keuls test).