Pharmacological examination of contractile responses of the guinea-pig isolated ileum produced by mu-opioid receptor antagonists in the presence of, and following exposure to, morphine

Abstract

We have assessed the potential of several mu-opioid receptor antagonists to elicit a response in the guinea-pig isolated ileum in the presence of, and following overnight exposure to, morphine. Naloxone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), (-)-5, 9alpha-diethyl-2-(3-furyl-methyl)-2'-hydroxy-6,7-benzomorphan (MR2266), but not D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), produced a transient inhibition of electrically-evoked contractions of the guinea-pig ileum. The effect of 1 microM CTOP, but not that to MR2266, was inhibited by 1 microM somatostatin. Naloxone (0.3 microM), CTOP (3 microM), CTAP (3 microM) and MR2266 (0.3 microM) antagonized the inhibitory effect of morphine on electrically-evoked contractions of the guinea-pig to a similar degree and, following 60 min exposure to morphine, produced non-sustained contractions. The response to 3 microM CTOP was significantly smaller than that to 3 microM CTAP. None of the antagonists produced a response in the absence of morphine. Following overnight exposure of the ileum to 0.3 microM morphine (4 degrees C), and repeated washing to remove the agonist, all four antagonists elicited non-sustained contractions. However, the responses to 3 microM CTOP and 0.3 microM MR2266 were significantly smaller than those elicited by 0.3 microM naloxone and 3 microM CTAP. Somatostatin (1 microM) significantly reduced naloxone-induced contractions, but not those to CTAP. While all four mu-opioid antagonists elicited contractions in the presence of, and following prolonged exposure to, morphine, differences between them were noted which may be a consequence of non-opioid actions.

Figure 1

The effect of morphine and naloxone on the guinea-pig isolated ileum. (a) A graph of electrically-evoked contractions (open columns) in the absence of morphine, the presence of 3 μM morphine and a combination of 3 μM morphine and 1 μM naloxone. Also shown is the withdrawal contraction (closed column) observed upon addition of 1 μM naloxone. Responses have been expressed as a percentage of the contraction to 60 mM KCl and shown as the mean±s.e.mean of nine (electrically-evoked contractions) and eight out of 9 (withdrawal contractions) observations. (b). A representative digitized trace recording of the effect of 1 μM naloxone on ‘fresh' segments of guinea-pig isolated ileum in the presence (upper, morphine-present) and absence (lower, morphine-naive) of 3 μM morphine. Note that 1 μM naloxone only produced a withdrawal contraction in the presence of morphine and that this was associated with a reversal of the inhibitory effect exerted by the agonist on electrically-evoked contractions. (c) A graph of electrically-evoked contractions (open columns) of preparations previously exposed to 3 μM morphine overnight (4°C), and subsequently washed to remove the agonist, in the absence and presence of 1 μM naloxone. Also shown is the withdrawal contraction (closed column) observed upon addition of 1 μM naloxone. Responses have been expressed as a percentage of the contraction to 60 mM KCl and shown as the mean±s.e.mean of seven (electrically-evoked contractions) and seven out of seven (withdrawal contractions) observations. (d). A representative digitized trace recording of the effect of 1 μM naloxone on segments of the guinea-pig isolated ileum previously stored overnight (4°C) in either the presence (upper; morphine-exposed) or absence (lower; morphine-naive) of 3 μM morphine, and subsequently washed repeatedly to remove the agonist. Note that 1 μM naloxone only produced a withdrawal contraction in the preparation previously exposed to morphine, but that this was not associated with a change in the magnitude of the electrically-evoked contractions.

Figure 2

A graph of the relationship between morphine concentration and the magnitude of withdrawal contraction of the guinea-pig isolated ileum elicited by naloxone. Preparations of the guinea-pig ileum were exposed to various concentrations of morphine and then exposed to 1 μM naloxone (‘fresh') or stored overnight (4°C) in the presence of morphine, washed repeatedly to remove the agonist, and challenged with 1 μM naloxone (overnight-morphine-exposed). Each preparation was exposed to only a single concentration of morphine and naloxone. Responses have been expressed as a percentage of the contractions to 60 mM KCl and shown as the mean±s.e.mean of the preparations that responded to naloxone. The number of experiments conducted are given in parenthesis: fresh, 0.03 μM (17), 0.1 μM (eight), 0.3 μM (17) and 3 μM (nine) morphine; overnight, morphine-exposed, five experiments for each concentration.

Figure 3

A comparison of the effect of morphine on electrically-evoked contractions of the guinea-pig isolated ileum following overnight storage (4°C) in either the absence (‘morphine-naive') or presence of 1 μM morphine (‘morphine-exposed') and subsequently washed repeatedly to remove the agonist. The electrically-evoked contractions have been expressed as a percentage of the response prior to the addition of morphine and are shown as the mean±s.e.mean of eight observations.

Figure 4

Comparison of the effect of (a) naloxone, (b) MR 2266, (c) CTOP and (d) CTAP on morphine-induced inhibition of electrically-evoked contractions of the guinea-pig isolated ileum. The electrically-evoked contractions have been expressed as a percentage of the response prior to the addition of morphine and are shown as the mean±s.e.mean of 5–10 observations.

Figure 5

Schild plot of the effect of various opioid receptor antagonists on morphine-induced inhibition of electrically-evoked contractions of the guinea-pig isolated ileum. Each log (DR-1) value represents the mean±s.e.mean of 5–10 observations.

Figure 6

Representative digitized recordings of the effect of (a) 3 μM CTOP, (b) 1 μM somatostatin (SS) and (c) 3 μM CTOP in the presence of 1 μM somatostatin (stippled bar), on electrically-evoked contractions of the guinea-pig isolated ileum. Note that CTOP and somatostatin caused a transient inhibition of electrically-evoked contractions but that CTOP failed to do in the presence of somatostatin.

Figure 7

A comparison of equieffective concentrations of the opioid receptor antagonists to elicit withdrawal contractions of the guinea-pig isolated ileum (a) in the presence of 0.3 μM morphine and (b) following overnight exposure (4°C) to 1 μM morphine and subsequently washed repeatedly to remove the agonist. Responses have been expressed as a percentage of the contraction to 60 mM KCl and shown as the mean±s.e.mean of eight (a) and 12 (b) observations. ^*Denotes a statistically significant difference from the effect of 3 μM CTAP.

Figure 8

Comparison of withdrawal contractions of the guinea-pig isolated ileum elicited by 0.3 μM naloxone (Nal) and 3 μM CTOP in the presence and absence of 1 μM somatostatin (SS). All preparations were exposed to 0.3 μM morphine prior to the addition of the antagonists. Responses have been expressed as percentage of the contraction to 60 mM KCl and shown are the mean±s.e.mean of eight observations. ^*Denotes a statistically significant difference (Mann Whitney U-test, P<0.05) for responses in the absence and presence of 1 μM somatostatin.