Pharmacological characterization of opiate physical dependence in the isolated ileum of the guinea-pig.

1 Physical dependence was produced in ilea from naive guinea-pigs by exposure of the tissue to different opiates for logarithmically-spaced periods of time (20-320 min). The responsiveness of the tissue to naloxone, as indicated by a strong contracture of the ileum, was enhanced in contrast to that found in intestines not exposed to opiates. 2 The dose-response curves to naloxone obtained in tissues individually exposed to different opiates showed that their relative potency in increasing sensitivity to naloxone was as follows: levorphan greater than morphine greater than Met-enkephalin greater than nalorphine greater than pentazocine. 3 The naloxone-induced response was dose-dependent and was directly related to the opiate concentration and length of exposure. 4 Dextrorphan the inactive isomer of levorphan, did not increase the responsiveness of the tissues to the narcotic antagonist, indicating that the phenomenon is stereospecific. 5 The naloxone-induced contraction in ilea exposed for 320 min to morphine (1 x 10(-6)M) was not prevented or suppressed by the administration of a large dose of morphine (1 x 10(-5)M) before or immediately after the naloxone challenge. 6 The evidence presented here shows that a phenomenon resembling in vivo opiate physical dependence can be acutely produced in vitro with pharmacological characteristics similar to other naloxone-induced abstinence effects.     

Clonidine dependence in the guinea-pig isolated ileum

1 Compared with the response of preparations incubated in solutions without clonidine, a three to four fold increase in the magnitude of the contracture of the longitudinal muscle to challenge with phentolamine (1.0 μm) was induced by incubating the guinea-pig isolated ileum at 22°C for 24 h with clonidine (1.0 μm) in Krebs solution containing hexamethonium (70 μm). Incubation of the ileum with clondine (1.0 μm) for 0.5 h at 37°C did not increase responsiveness to phentolamine.2 The increase in responsiveness to phentolamine was directly related to the clonidine concentration in the incubation fluid over the range 0.01 to 1.0 μm.3 The magnitude of the contracture to phentolamine of ilea incubated with clonidine (1.0 μm) (withdrawal contracture) was directly related to the challenge dose of phentolamine over the range 0.3 to 1.0 μm.4 Yohimbine (1.0 μm) or piperoxane (1.0 μm) elicited a response comparable to that elicited by phentolamine but propranolol (1.0 μm) was inactive.5 Addition of phentolamine (1.0 μm) to clonidine (1.0 μm) in the incubation fluid abolished the increased response of the preparation to subsequent challenge with phentolamine.6 Addition of hyoscine (0.5 μm) immediately after challenge with phentolamine restored the tension of the withdrawal contracture to its resting level.7 Tetrodotoxin (3.0 μm) given before challenge, prevented phentolamine from eliciting a withdrawal contracture.8 Ileal segments incubated with clonidine (1.0 μm) were unresponsive to challenge with naloxone (100 nm); and segments incubated with normorphine (1.0 μm) were unresponsive to phentolamine (1.0 μm), although responsive to naloxone.9 Normorphine (1.0 μm) restored to resting level the tension of the clonidine withdrawal contracture; and clonidine (0.1 μm) restored to resting level the tension of the contracture to naloxone in ileal segments incubated with normorphine.10 These experiments indicate that incubation with clonidine induces, in the final cholinergic motor neurones of the myenteric plexus of the isolated ileum, a dependence the withdrawal from which is expressed as a contracture in response to α-adrenoceptor antagonists.11 Although opiate receptors are not involved in clonidine dependence nor α-adrenoceptors in opiate dependence, the findings that normorphine suppresses the clonidine withdrawal-contracture and that clonidine suppresses the contracture of opiate-dependent ileum to naloxone, suggest that the withdrawal effect studied in both clonidine and normorphine dependence in this preparation is mediated by release of acetylcholine from the final motor neurone.     

Peristalsis in the isolated guinea-pig ileum during opiate withdrawal

Summary Naloxone enhances peristalsis in isolated intestinal segments from morphine-dependent guinea-pigs to a greater degree than in naive preparations. These data suggest that a peripheral mechanism may be involved in the expression of opiate withdrawal in the small intestine, and indicate that the action of this mechanism leads, at least in vitro, to an increase in the number of peristaltic waves.     

Opioid dependence and cross-dependence in the isolated guinea-pig ileum

The development of opioid dependence and tolerance attributed to selective types of opiate receptors was studied in the isolated ileum of guinea pigs chronically exposed to specific opioids. These investigations were based on reports that in this preparation highly tolerant opiate receptors may coexist with opiate receptors of almost unchanged sensitivity. Thus, the ilea were set up in vitro and tested for tolerance and dependence. Apparently precipitation of the withdrawal contracture, indicating dependence, proved a more sensitive parameter than the phenomenon of tolerance. Maximal dependence was determined at rather low degrees of tolerance (5 to 10 fold). The intensity of the withdrawal contracture failed to increase as opiate tolerance did. Furthermore, the experiments failed to present evidence for the existence of selective dependence at specific opiate receptor types. These findings may suggest multiple adaptational mechanisms upon chronic activation of opiate receptors. One mechanism may be responsible for the development of dependence and a low degree of tolerance, whilst a further increase of tolerance may be associated with changes at the opiate binding site level.     

丹参提取物对豚鼠回肠体外吗啡依赖性的影响

目的观察丹参脂溶性抗吗啡依赖有效成分SmE 2对吗啡依赖离体豚鼠回肠纳洛酮催促后收缩性的影响,并观察丹参提取物自身潜在的依赖性,最后对丹参有效成分SmE 2进行初步鉴定.方法采用豚鼠离体回肠吗啡依赖模型,实验分为空白对照组、吗啡依赖组、SmE2给药组和SmE2自身依赖组,观察各组离体豚鼠回肠的收缩状况;建立高效液相色谱法(HPLC)鉴定丹参主要脂溶性的成分.结果与吗啡依赖组比较,0.05,0.25,1.25 g·L-1SmE2预先孵育吗啡依赖离体豚鼠回肠可剂量依赖性地减弱纳洛酮催促后的特异性戒断收缩;高剂量SmE2(1.25 g·L-1)长时间孵育后,不引起离体豚鼠回肠纳洛酮催促产生的特异性戒断收缩;经HPLC法初步鉴定丹参主要脂溶性有效成分为隐丹参酮.结论SmE2能在一定程度上抑制吗啡依赖离体豚鼠回肠的戒断性收缩,且自身无潜在身体依赖性;SmE2主要成分为隐丹参酮.     

A possible role for prostaglandins in the expression of morphine dependence in guinea-pig isolated ileum.

1. The effects of the cyclo-oxygenase inhibitors, indomethacin (1.3 microM) and mefenamic acid (10 microM), were investigated on the naloxone-precipitated withdrawal contracture of the morphine-dependent guinea-pig ileum in vitro. 2. Both indomethacin and mefenamic acid prevented expression of the withdrawal contracture on naloxone challenge. 3. The effects of indomethacin were reversed by the concurrent application of a low dose of prostaglandin E1 (10 nM). 4. It was concluded that prostaglandins probably play a role in the expression of morphine withdrawal in this model.     

Action of bretylium on the isolated guinea-pig ileum

Bretylium inhibits the contractions of the longitudinal muscle caused by histamine and, to a lesser extent, those by acetylcholine, carbachol and nicotine. It is a very weak antagonist of 5-hydroxytryptamine and in concentrations of up to 190 μg/ml. does not inhibit the actions of bradykinin and substance P on the longitudinal muscle. The inhibitory effect of bretylium on the emptying phase of the peristaltic reflex is similar to that of ganglion blocking agents, while the much less pronounced effect on the contraction of the longitudinal muscle during the preparatory phase is assumed to be due to a weak atropine-like action.     

Arachidonic acid and its metabolites are involved in the expression of morphine dependence in guinea-pig isolated ileum

The effects of phospholipase A₂, cyclooxygenase-1, cyclooxygenase-2 and 5-lipoxygenase inhibitors were investigated on the naloxone-precipitated withdrawal contracture of the acute morphine-dependent guinea-pig ileum in vitro. Mepacrine (a phospholipase A₂ inhibitor), tolmetin (selective cyclooxygenase-1 inhibitor) and meloxicam (selective cyclooxygenase-2 inhibitor) treatment before or after morphine was able to both prevent and reverse the naloxone-induced contracture after exposure to morphine in a concentration-dependent fashion. Also, nordihydroguaiaretic acid (5-lipooxygenase inhibitor) was able to block the naloxone-induced contracture following exposure to morphine when injected before or after the opioid agonist. The results of the present study suggest that arachidonic acid and its metabolites (prostaglandins and leukotrienes) are involved in the development of opioid withdrawal.     

Differential influence of D1 and D2 dopamine receptors on acute opiate withdrawal in guinea-pig isolated ileum

1. The effects exerted by D₁ and D₂ dopamine agonists and antagonists on the acute opiate withdrawal induced by μ- and κ-receptor agonists were investigated in vitro.
2. Following a 4 min in vitro exposure to morphine (moderately selective μ-agonist), [D-Ala², Me-Phe⁴, Gly-ol⁵]enkephalin (DAMGO, highly selective μ-agonist) or U-50488H (highly selective κ-agonist) the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone.
3. The non-selective dopamine receptor antagonist haloperidol when added before or after the opioid agonists, was able dose-dependently to prevent or to reverse the naloxone-induced contracture after exposure to μ- (morphine and DAMGO) and κ- (U-50488H) opioid agonists. The non-selective dopamine receptor agonist, apomorphine, was able to exert the same effects only at the highest concentration used.
4. The selective D₂ dopamine receptor antagonist, sulpiride, was also able to reduce dose-dependently both μ- and κ-opioid withdrawal, whereas the D₁-receptor selective antagonist SCH 23390 did not affect either μ- or κ-opioid withdrawal.
5. Bromocriptine, a D₂ selective dopamine receptor agonist was able to increase significantly, and in a concentration-dependent manner, the naloxone-induced contracture by μ- and κ-opioid agonists, whereas SKF 38393, a D₁ selective dopamine receptor agonist, increased only the withdrawal after morphine or U50-488H.
6. Our data indicate that both D₁ and D₂ dopamine agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the dopaminergic system and opioid withdrawal at both the μ- and κ-receptor level.
7. Furthermore, the ability of sulpiride to block strongly opiate withdrawal when compared to SCH 23390, as well as the effect of bromocriptine to increase opiate withdrawal suggest that D₂ dopamine receptors may be primarily involved in the control of opiate withdrawal.

     

Calcium dependence of baclofen- and GABA-induced depression of responses to transmural stimulation in the guinea-pig isolated ileum

Both baclofen and gamma-aminobutyric acid (GABA) induced a dose-dependent depression of cholinergic twitch contractions in the transmurally stimulated guinea-pig isolated ileum. Over a range of 0.6-2.4 mM Ca2+, the degree of depression was inversely related to the Ca2+ concentration, with an increased sensitivity and sinistral shift of the dose-response curve at lower Ca2+ concentrations. Partial occupation of Ca2+ channels by Ruthenium Red (0.1 microM) also potentiated the depressive responses to baclofen and GABA. It is concluded that these agonists, acting through GABAB receptors, limit the availability of Ca2+ required for neurotransmitter release in myenteric motor nerves.     

Anticholinergic properties of progesterone in the isolated ileum of the guinea-pig

To determine if the cholinergic system is involved in the intestinal smooth muscle relaxant action of progesterone, segments of guinea-pig ileum were mounted in 20 ml chambers containing Krebs solution. In one group of experiments, we studied the effect of various doses of progesterone and atropine on the twitch-like contraction of the ileum caused by electrical stimulation. In the second group of experiments, dose-response curves were constructed for acetylcholine, histamine, and barium chloride alone and in the presence of progesterone or atropine. Progesterone, like atropine, decreased in a dose-dependent manner the phasic contractions and tone of the unstimulated intestinal segments, and exerted a rapid, reversible, and dose-dependent inhibitory action on intestinal responses to electrical stimulation. In addition, in the presence of the hormone or atropine, the dose-response curve to acetylcholine was clearly shifted to the right in a parallel manner with no change in maximal response. At the concentrations tested, neither progesterone nor atropine blocked the contractile effect of barium chloride on the ileum and only slightly reduced the spasmogenic action of histamine. These results suggest that the relaxant action of progesterone on the intestinal smooth muscle may be mediated through muscarinic receptors, indicating that progesterone is an endogenous product with anticholinergic activity. © 1996 Wiley-Liss, Inc.     

Tetrahydroisoquinolines (TIQs) do not act on opiate receptors in the guinea-pig ileum

The myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum was stimulated with supra-maximal electrical field stimulation at 0.1 Hz. The contractile response was inhibited by salsolinol (1-300 microM) and tetrahydropapaveroline (THP) (3-30 micro M) but not by (cis)-3-carboxysalsolinol. S(-)-Salsolinol was more potent than R(+)-salsolinol. The inhibition by salsolinol and THP was unchanged by naloxone (up to 10 micro M). However, naloxone completely prevented the inhibition induced by normorphine, with a pA2 of 8.66. The results indicate that salsolinol and THP do not interact with opiate receptors in this preparation.     

Opioid dependence in myenteric neurons innervating the circular muscle of guinea-pig ileum

Summary Guinea-pigs were treated with morphine for 6–8 days by subcutaneous implantation of pellets, each containing a mixture of morphine base (120 mg) and morphine hydrochloride (35 mg). Each guinea-pig received a single pellet. Mechanical activity of the circular muscle was recorded in vitro in preparations comprising the circular muscle and myenteric plexus. Exposure to morphine was maintained by addition of 1 M morphine to the organ baths. After 90 min, morphine was withdrawn, either by repeatedly washing tissues in morphine-free Krebs' solution , or by addition of naloxone to reduce the occupancy of the opioid receptors by morphine. Withdrawal of morphine resulted in markedly enhanced contractile activity compared with that in circular muscle-myenteric plexus preparations from untreated control guinea-pigs. The withdrawal contractions were abolished by tetrodotoxin (600 nM) and greatly reduced by hyoscine (1 M), indicating that they resulted from action potential discharge in myenteric neurons that release acetylcholine onto the circular muscle. Activation of the cholinergic excitatory motor neurons was not secondary to synaptic activation by cholinergic interneurons, because hexamethonium (100 M) did not affect withdrawal contractions. The withdrawal response may therefore arise in the cholinergic excitatory motor neurons themselves, or in neurons that activate them via noncholinergic mechanisms. Send offprint requests to S. Johnson at the above address     

Contractile effect of (+)-glaucine in the isolated guinea-pig ileum.

The intestinal effects of (+)-glaucine [(S)-1,2,9,10-tetramethoxyaporphine] were studied using the guinea-pig ileum. (+)-Glaucine (10-300 microM) induced ileal contractions. The contraction was not affected by tetrodotoxin, atropine, hexamethonium, propranolol, naloxone, methysergide, N(G)-nitro-L-arginine methyl ester, SR141716A (a cannabinoid CB1 receptor antagonist) or SR140333 (a tackykinin NK1 receptor antagonist) plus SR48968 (a tackykinin NK2 antagonist). (+)-Glaucine-induced contraction was reduced by indomethacin, nordihydroguaiaretic acid or bisindolylmaleimide I and abolished by verapamil and nifedipine. These results suggest that (+)-glaucine-induced contraction involves activation of voltage-dependent Ca2+ channels and protein kinase C and could be mediated by the release of arachidonic acid metabolites.     

The biphasic response of the isolated guinea-pig ileum by bradykinin

The isolated guinea-pig ileum, challenged by agonist, was used to study the effect of bradykinin. In the presence of acetylcholine producing approximately 60% of maximum contraction, bradykinin caused relaxation followed by contraction. The biphasic response to bradykinin was also found in the presence of histamine, eledoisin, angiotensin, prostaglandin F_2α and transmural electrical stimulation. The conditions for bradykinin-induced relaxation were not found after treatment by bradykinin, and potassium or barium chloride. Under conditions where bradykinin produced a biphasic response, acetylcholine, histamine, eledoisin, angiotensin, prostaglandin F_2α and lysine-vasopressin only contracted the ileum, while adrenaline, noradrenaline, oxytocin, calcium and magnesium chloride only relaxed. On increasing the percentage of maximum contraction with acetylcholine, an inverse relationship with relaxation by bradykinin was found. Tachyphylaxis was not present with the bradykinin-induced relaxation. The relaxing effect of bradykinin is more likely to be due to a direct action on the muscle cell membrane than to a release of a mediator or to blockade of a receptor mediating contraction.     

Cortisone: a potent GABAA antagonist in the guinea-pig isolated ileum

In the guinea-pig isolated ileum, cortisone at 0.001-10 nM induced a non-competitive, dose-dependent antagonism of GABAA-receptor-mediated contractile responses to applied GABA, depressing the maximum contractile response to GABA (100 microM), without affecting contractile responses to acetylcholine or cholinergic twitch contractions. At higher concentrations (greater than 10 nM), cortisone depressed contractile responses to acetylcholine (10-100 nM) and cholinergic twitch responses to transmural stimulation. Cortisone is thus the most potent non-competitive antagonist at GABAA-receptor complexes in the guinea-pig ileum. From molecular modelling, sterically there appeared little difference between cortisone and cortisol, the latter being an enhancer of GABAA-receptor-mediated action in the ileum. However, there were significant differences in electrostatic potentials between the two steroids, due to the different levels of oxidation at C11 which may contribute to such opposing actions.     

Evidence for two leukotriene receptor types in the guinea-pig isolated ileum

Leukotriene (LT) receptors in the guinea-pig ileum were characterized using LTB4, LTC4, LTD4 and LTE4 and the LT antagonists FPL 55712, ICI 198615 and (+/-)SKF 104353. LTB4 was inactive but the other LTs induced concentration-related contractions. LTC4 responses differed to those induced by LTD4 or LTE4. Inhibitors of LT metabolism had no significant effects on any LT responses. LTD4 contractions were inhibited by all three antagonists but a resistant response was apparent at concentrations of ICI 198615 greater than 10(-8) M. All three antagonists were weak/inactive against LTC4. LTE4 was a partial agonist which antagonized LTD4 responses but had little or no activity against LTC4 or histamine. These results suggest that two distinct LT receptor types exist on guinea-pig ileum. One type is predominantly activated by LTD4 and is antagonized by three structurally distinct LT antagonists and the partial agonist LTE4. The second type is predominantly activated by LTC4 and is insensitive to the LT antagonists.