Multiple opiate receptors in guinea-pig ileum

Actions of the prototypic μ-, κ-, and σ-opiate receptor agonists, morphine (M) ketocyclazocine (K) and SKF-10,047. (S), respectively, were examined and differentiated using the guinea-pig ileum preparation. S, like M and K, depressed the electrically stimulated ileum. Naloxone antagonized the depressant actions of the prototypic drugs with different potencies. PA₂ values of naloxone for M, K, and S, respectively, were 8.81, 7.58 and 7.74. Relative cross tolerance of each prototypic drug to normorphine, a comparison standard, was also examined in morphine-pretreated ilea and quantitatively estimated as follows: (1) the median effective dose of each drug and of the standard drug normorphine were determined in the nontolerant ileum (IC50_NT) and in ilea with varying degrees of tolerance IC50_T); (2) cross-tolerance ratios (IC50_T/IC50_NT) of each drug and of normorphine were calculated for the varying degrees of tolerance; (3) cross-tolerance ratios of each drug were plotted against those of normorphine, the data were fit by a least squares straight line, and the slope of the line determined as the Relative Cross Tolerance Index (RCTI). RCTI for M was 2.21. K and S, however, had lower RCTI's of 0.44 and 0.64 respectively. In the morphine-pretreated tolerant ilea, slopes of the dose response curves of the prototypic drugs were found to differ: while M and K possessed steep and constant slopes for ilea with different degrees of tolerance, the slopes for S became shallower as ilea became more tolerant to morphine. A maximum ceiling effect of less than 50% depression was obtained for S in the most highly tolerant ilea. The above observations are consistent with possible existence of the three types of hypothesized opiate receptors in the guinea-pig ileum.     

Aspects of opiate dependence in the myenteric plexus of the guinea-pig.

Myenteric plexus-longitudinal muscle strips prepared from tolerant/dependent guinea-pigs and continuously exposed to normorphine, display a contracture upon naloxone challenge. This phenomenon represents a sign of abstinence. Removal of the opiate by extensive washing resulted in the failure of naloxone to induce the abstinence sign, while the plexus still displayed considerable, although reduced, tolerance to morphine. Reexposure of withdrawn preparations to normorphine reinduced the ability to display the abstinence sign. Highly tolerant preparations exhibited a 30 fold increase in sensitivity to serotonin and prostaglandin E₁ when tested a few minutes after naloxone-precipitated withdrawal. Supersensitivity rapidly declined when normorphine was washed off the preparation, while reincubation of withdrawn tissues with the opiate resulted in reinduction of supersensitivity. The data confirms a close relationship between a state of tolerance and dependence (including display of the abstinence sign) and supersensitivity to putative neurotransmitters or neuromodulators, becoming evident following administration of naloxone.     

Reduction in opiate receptor reserve in morphine tolerant guinea pig ilea

Spare opiate receptors in the guinea pig ileum have been detected by the use of the opiate receptor alkylating agent beta-chlornaltrexamine (CNA). Treatment of the guinea pig ileum longitudinal muscle in vitro with low concentrations (less than 10nM) of CNA resulted in an irreversible parallel shift to the right of the normorphine log concentration response curve. With increasing concentration of the reagent, the agonist EC50 becomes progressively greater. Finally a point is reached at which the maximal agonist effect decreases, so that parallelism is no longer seen. The maximal parallel shift provides a measure from which one can estimate the spare receptor fraction that is present in untreated tissue. In ilea from normal guinea pigs, roughly 80-90% of the opiate receptors for normorphine were found to be spare. Even after the largest parallel shifts that could be achieved, the naloxone Ke value for antagonism was unchanged, indicating that normorphine acts through spare mu receptors. Ilea from guinea pigs made tolerant by chronic morphine pellet implantation were found to be more sensitive to the effects of CNA treatment; there was a reduction in the number of spare receptors for normorphine. It is suggested that the opiate spare receptor fraction is physiologically modulated to control neuronal sensitivity to opioid effect.     

Application of an irreversible opiate antagonist (β-FNA, β-funal-trexamine) to demonstrate dynorphin selectivity for K-opioid sites

Application of 100 nM β-FNA for 60 minutes to isolated longitudinal muscles-myenteric plexus preparations from the guinea pig ileum caused a marked antagonism of the inhibitory action of normorphine and leucine enkephalin without greatly affecting the inhibitory potency of dynorphin or ethylketocyclazocine. The interaction of β-FNA with the normorphine (μ-opiate receptors) appears to be non-equilibrium. Pretreatment with β-FNA caused a significant increase in the apparent naloxone dissociation constant for normorphine and leucine enkephalin but not for dynorphin or ethylketocyclazocine. The results lend further support to the hypothesis that normorphine and the enkephalins activate preferentially μ-opiate receptors on the ileum, whereas dynorphin interacts predominantly at k-opiate sites.     

Morphine dependence in the isolated guinea-pig ileum and its modification by p-chlorophenylalanine.

Experiments were performed to quantitatively determine morphine physical dependence in the isolated guinea-pig ileum and to assess the influence of p-chlorophenylalanine (PCPA) on its development. Ileum segments taken from animals treated with 10 s.c. injections of 100 mg/kg of morphine, given at intervals of 8 hr without interruption, responded with intense, prolonged, dose-dependent contractions to the $\text{in}$$\text{vitro}$ administration of naloxone, although contractions guinea-pigs also responded to naloxone, although contractions were smaller and of short duration. The sensitivity to naloxone on segments isolated from morphinized animals was compared to that of controls. Ilea from morphine-treated guinea-pigs were 8 to 32 times more sensitive to naloxone, as determined by a shift in the naloxone concentration-response curve to the left. There was also a three-fold increase in the maximum response. This phenomenon was taken as evidence of narcotic dependence. PCPA, given before morphine administration, at doses producing only a slight (11%) decrease in intestinal serotonin (5-HT) levels, partially reduced the sensitivity of the morphine-treated ileum to naloxone. However, high doses of PCPA, decreasing intestinal 5-HT by 40%, enhanced the abstinence-like effects of naloxone in the morphine pretreated ileum. PCPA by itself changed the responsiveness of the non-morphinized ileum to naloxone. The direction and magnitude of the change produced by PCPA alone was roughly equivalent to that produced by the serotonin depletor in the morphinized ileum. This finding indicates that PCPA has no effect upon the development of physical dependence in the isolated ileum. It remains to be determined whether or not the increased sensitivity to naloxone induced by high doses of PCPA has something in common with the changes in responsiveness to the antagonist induced by narcotics.     

The development of opiate tolerance may dissociate from dependence

Experiments on opiate sensitive peripheral tissue preparations such as the mouse vas deferens and the guinea-pig ileum have demonstrated the ability to induce very high degrees of selective tolerance towards particular opiate agonists. Interestingly, the highly tolerant mouse vas deferens failed to display any sign of dependence as judged by the inability of naloxone to precipitate a withdrawal sign. In analogy, the present studies on the guinea-pig ileum revealed a striking dissociation in the degree of tolerance and dependence developed. Opiate receptor binding studies on both tissues point to distinct differences in the opiate-induced effector mechanisms. It is concluded that adaptational changes upon chronic opiate receptor activation may occur at multiple sites within the effector system of the opiate receptor.     

Latency to first naloxone-induced jump as a measure of precipitated abstinence intensity in morphine-dependent mice and the interaction of set on this test

A method developed in this laboratory uses latency to time of first jump after injection of naloxone rather than the number of jumps in a specified period or the number of animals jumping as a measure of the degree of morphine physical dependence. For the test, mice are placed in a glass cylinder used as a test chamber, after being injected with the antagonist. During the development of this method it was observed that repeated exposures of dependent mice to both naloxone and the chamber yielded shorter latencies to first jump than did repeated exposures to naloxone alone in animals with the same degree of physical dependence. It appears that learning develops when naloxone injections are given repeatedly and followed by exposure to the test chamber and that this learned behavior is manifested by a reduced latency to first jump which may be confused with increased intensity of the opiate-withdrawal syndrome.     

Response to opioids of isolated small intestine in the house musk shrew, Suncus murinus.

1. Response to the opioids of isolated small intestine of Suncus murinus, an insectivore, was examined and compared with that of guinea-pig ileum. 2. The mechanical response to morphine and U50,488H, preferential mu- and kappa-agonist respectively, was relaxation which was antagonized by tetrodotoxin and naloxone in the Suncus small intestine. 3. Methionine5-enkephalin and D-alanine2, D-leucine5-enkephalin, both preferential delta-agonists, elicited contraction or relaxation in the Suncus small intestine, while enkephalins elicited sustained contraction in the tetrodotoxin-pretreated intestine. 4. In the guinea-pig ileum, the response to morphine and enkephalins was consistently relaxation which was antagonized by tetrodotoxin and naloxone. 5. Morphine, D-alanine2,D-leucine5-enkephalin and U50,488H inhibited electrically-evoked twitch responses of intestine in a concentration-dependent manner in both animals. 6. The relative potencies calculated from IC50 values of opioids for twitches followed the order, D-alanine2,D-leucine5-enkephalin greater than U50,488H greater than morphine in the Suncus small intestine, whereas U50,488 greater than D-alanine2,D-leucine5-enkephalin greater than morphine in the guinea-pig ileum. 7. Suncus small intestine has unique features such that D-alanine2,D-leucine5-enkephalin was most potent in inhibiting electrically-evoked twitches and that enkephalins induced contraction following tetrodotoxin pretreatment.     

Tyr-MIF-1 and hemorphin can act as opiate agonists as well as antagonists in the guinea pig ileum.

The brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was tested for its effects on electrically stimulated contractions in the guinea pig ileum assay. Tyr-MIF-1 acted as an opiate agonist in reducing these contractions. Its IC50 was about 9 microM, and its effects were reversed by naloxone and CTOP. The ability of Tyr-MIF-1 also to antagonize the inhibitory effects of opiates on electrically stimulated contractions was more evident in the ileum removed from a guinea pig tolerant to morphine or after partial inactivation of opiate receptors with beta-CNA. Similar results were observed with hemorphin. The endogenous peptide Tyr-MIF-1 and the blood-derived peptide hemorphin, therefore, can act as agonists as well as antagonists in the guinea pig ileum. The effects as antagonists are best observed in preparations of ileum with reduced receptor reserve (tolerant or beta-CNA treated) and are consistent with the idea that properties of endogenous peptides as opiate antagonists are enhanced in the tolerant state.     

Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia,tolerance and physical dependence

The findings from our laboratory indicated that pharmacological manipulations of GABA system modified morphine analgesia, tolerance and physical dependence. Elevating brain levels of GABA by slowing its destruction with aminooxyacetic acid not only antagonized the analgesic action of morphine in both non-tolerant and tolerant mice, but also enhanced the development of tolerance and physical dependence. On the other hand, blockade of postsynaptic sites of GABA receptors by bicuculline resulted in an inhibition of tolerance and dependence development. Administration of 2,4-diaminobutyric acid, an inhibitor of GABA uptake in the neurons, antagonized morphine analgesia in both non-tolerant and tolerant mice. However, it did not modify naloxone precipitated withdrawal jumping. On the contrary, β-alanine, an inhibitor of the GABA uptake process in glial cells, potentiated naloxone precipitated withdrawal jumping in morphine dependent mice, but it had no effect on morphine antinociception in both non-tolerant and tolerant mice.     

Comparison of physical dependence of ohmefentanyl stereoisomers in mice

Stereo-structural difference of ohmefentanyl stereoisomers on analgesic action and receptor affinity has been studied. To assess the difference of ohmefentanyl stereoisomers in physical dependence, the potency of physical dependence was quantified by estimating the ED50 value of ohmefentanyl stereoisomers in the naloxone-precipitated jumping test in mice. Morphine was used to assess the method and as a drug of comparison. The results indicate that the degree of physical dependence of morphine can been quantified by estimating the ED50 value of morphine withdrawal jumping induced by naloxone. A significant difference was observed in withdrawal jumping ED50 values among ohmefentanyl stereoisomers. Of these isomers, F9202 and F9204 had similarly potent analgesic action, but very significant difference in naloxone precipitated withdrawal response. Dependent potency index of F9204 was 618-fold weaker than that of F9202. It is concluded that a stereo-structural difference in physical dependence is found to exist among ohmefentanyl stereoisomers. Compound F9204 displayed a strong analgesic action and weak physical dependent potency.     

Finasteride, a 5alpha-reductase inhibitor, potentiates antinociceptive effects of morphine, prevents the development of morphine tolerance and attenuates abstinence behavior in the rat

It has been shown that morphine increases 5alpha-reductase enzyme activity in the rat central nervous system; however importance of this finding on morphine analgesia, tolerance and dependence has not been reported. In the present study, we investigated inhibition of 5alpha-reductase enzyme on morphine effects using finasteride. To determine whether the 5alpha-reductase enzyme interact with morphine analgesia, finasteride (5 mg/kg, i.p.) was administrated with morphine (5 and 7 mg/kg, i.p.). The tail-flick test was used to assess the nociceptive threshold, before and 15, 30, 45, 60 and 90 min after drug administration. In tolerance experiments, morphine 20 mg/kg was injected i.p., twice daily for 4 days. The development and expression of dependence were assessed in the naloxone precipitation test 5 days after the morphine (20-30 mg/kg, i.p.) administration. We found that finasteride could potentiate the antinociceptive effect of morphine. In addition, chronic finasteride administration effectively blocked development of tolerance and dependence to morphine. Following chronic morphine administration, single dose injection of finasteride failed to reverse tolerance but prevented naloxone precipitate withdrawal syndrome. Therefore, it was concluded that there is a functional relationship between 5alpha-reductase enzyme and morphine.     

Antinociceptive effects of morphine and naloxone in mu-opioid receptor knockout mice transfected with the MORS196A gene

Background  

Opioid analgesics such as morphine and meperidine have been used to control moderate to severe pain for many years. However, these opioids have many side effects, including the development of tolerance and dependence after long-term use, which has limited their clinical use. We previously reported that mutations in the mu-opioid receptors (MOR) S196L and S196A rendered them responsive to the opioid antagonist naloxone without altering the agonist phenotype. In MORS196A knock-in mice, naloxone and naltrexone were antinociceptive but did not cause tolerance or physical dependence. In this study we delivery this mutated MOR gene into pain related pathway to confirm the possibility of in vivo transfecting MORS196A gene and using naloxone as a new analgesic agent.     

RP 67580, a potent and selective substance P non-peptide antagonist]

The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.     

Fluctuations of acetylcholinesterase in the mouse spinal cord and in vivo sodium effect during the development of morphine tolerance,dependence, and withdrawal

Tolerance to and physical dependence on morphine were produced and assessed in Swiss inbred albino mice by giving morphine sulphate (s.c.) three times a day for a period of 15 days in an increasing dose of 10 mg/kg every 24 hours. Physical dependence was assessed taking naloxone induced jumping as well as weight loss during normal withdrawal into consideration. The effect of sodium ions in the potency of naloxone in antagonizing morphine's effect was also analyzed. The spinal cord was assayed for acetylcholinesterase employing both biochemical and histochemical parameters. It was found that the amount of the enzyme increased with the development of tolerance but the amount decreased as the animals became physically dependent. However, the values were significantly above the control. Administration of naloxone brought about a sudden and significant fall in the level of the enzyme. Normal withdrawal too was characterized by a weak activity of the enzyme. It has been found that sodium ions can influence naloxone antagonism in an in vivo system.     

Adenosine receptors are involved in the control of acute naloxone-precipitated withdrawal: in vitro evidence

The effects exerted by adenosine A1 and A2 receptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 adenosine receptor agonist, adenosine, was able to reduce dose-dependently naloxone-precipitaded withdrawal. The same effect was induced by the adenosine A1 receptor agonist, N6-Cyclopentyladenosine (CPA) whereas the selective adenosine A2A receptor agonist CGS 21680 increased the naloxone-precipitated withdrawal phenomenon. Dipyridamole, a blocker of adenosine reuptake, induced a significant reduction of morphine dependence. Caffeine, an adenosine receptor antagonist, significantly increased the naloxone-precipitated withdrawal effect in a concentration dependent manner. The same effect was observed with 8-phenyltheophylline (8PT), an A1 adenosine receptor antagonist, whereas 3,7-dimethyl-1-propargylxanthine (DMPX), an A2 adenosine receptor antagonist, reduced the naloxone-precipitated withdrawal phenomenon. The results of our experiments indicate that both A1 and A2 adenosine receptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the adenosine receptors and opioid withdrawal.     

(3R)-4-[(3R)-3-Amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC(50)=18nM) with 3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC(50)=2.6nM), selective, and efficacious in an oral glucose tolerance test in mice. It was selected for extensive preclinical development as a potential back-up candidate to sitagliptin.     

Opioid properties of beta-lipotropin fragment 60-65, H-Arg-Try-Gly-Gly-Phe-Met-OH.

The pharmacologic activity of the hexapeptide fragment corresponding to the amino acid fragment 60–65 in β-lipotropin, (β-LPH-(60–65)) was studied $\text{in vitro}$ and $\text{in vivo}$. In binding assays on synaptosomal plasma membrane the peptide was found to be equipotent to met-enkephalin, but behaved differently to cations; in contrast to met-enkephalin both Mn⁺² and Na⁺ enhanced the binding of β-LPH-(60–65) to synaptosomal plasma membrane. On both the quinea pig ileum and mouse vas deferens β-LPH-(60–65) inhibited contractions elicited by electrical stimulation and each effect was reversible by naloxone. On the guinea pig ileum β-LPH-(60–65) was equipotent to met-enkephalin and 0.5 as potent as normorphine but on the vas deferens it was 4.6 times more potent than normorphine. The activities of β-LPH-(60–65) appear to be due to the intact compound rather than to its conversion to met-enkephalin, since the peptide extracted from the ileum assay was found to behave identically as β-LPH-(60–65) with high pressure liquid chromatography. When β-LPH-(60–65) was administered centrally to mice and rats, no overt central actions were observed and an antinociceptive effect could not be demonstrated. Nor did β-LPH-(60–65) antagonize morphine action or precipitate the withdrawal syndrome in morphine dependent animals. It is concluded that the good agreement which generally exists between $\text{in vitro}$ and $\text{in vivo}$ assay procedures for opiate-like activity of morphine and its surrogates does not necessarily hold for the endogenous peptides with similar actions.     

The monoterpene alkaloid cantleyine from Strychnos trinervis root and its spasmolytic properties.

Cantleyine, a monoterpene alkaloid isolated from the root bark of Strychnos trinervis, was submitted to a broad spectrum pharmacological screening, in which the principal effect observed was a nonspecific relaxation of isolated smooth muscles. Cantleyine relaxed (IC50 2.1 x 10(-4) M) the guinea-pig trachea, pre-contracted by carbachol and antagonized in a nonspecific manner; carbachol (IC50 2.1 x 10(-4) M) and histamine (IC50 1.4 x 10(-4) M) induced contractions in the guinea-pig ileum; and phenylephrine (IC50 3.8 x 10(-4) M) responses in the rat aorta. Cantleyine antagonized (pD'2, 3.82) cumulative concentration response curves to histamine in the ileum in a noncompetitive, reversible (slope, 4.84) and concentration dependent manner. The tonic contractions induced by histamine and KCl were also inhibited in a concentration-dependent and reversible manner (IC50 7.2 x 10(-5) and 1.8 x 10(-4) M, respectively), suggesting that cantleyine should be acting on voltage-dependent Ca2+ channels. This hypothesis was confirmed by the observation that cantleyine inhibited (pD'2, 3.35), in a concentration dependent manner, the CaCl2 induced contraction in depolarizing medium. These results suggest that cantleyine produces nonspecific spasmolytic effects in smooth muscle and that in guinea-pig ileum this effect is, in part, due to the inhibition of Ca+2 influx through voltage-dependent Ca2+ channels.     

Differentiation of beta-adrenoceptors in right atrium, diaphragm and adipose tissue of the rat, using stereoisomers of propranolol, alprenolol, nifenalol and practolol.

2-Diazomorphine-bovine serum albumin (2-DAM-BSA) was prepared by diazotizing p-aminobenzoyl-BSA to morphine. Rabbits immunized with 2-DAM-BSA produced antibodies directed to morphine. A 50 percent reduction in ³H-morphine binding required 4.4 pmol of morphine, and 60, 225, and 350 pmol of normorphine, morphine-3-glucuronide, and codeine, respectively. A radioimmunoassay for brain morphine is described, validated, and used to determine if naloxone alters brain morphine in morphine pelleted mice. The apparent biological half-life of morphine in brain was approximately 52 hours between 24 and 72 hours after pellet implantation, and decreased to 1.25 hours after pellet removal. Naloxone (10 mg/kg) administered 24, 48, or 72 hours after implantation and in doses of 1.0–100 mg/kg administered at 48 hours resulted in either no significant change, or, in a few experiments, increased the brain concentration of morphine. The present experiments could not detect a fraction of total brain morphine that is reduced by naloxone.