Characterization of kappa-opioid receptors in the guinea-pig ileum

Equilibrium binding saturation studies with [3H]bremazocine, under mu- and delta-suppressed conditions and [3H]U69593 have demonstrated that both radioligands bind with high affinity to an apparently homogeneous population of binding sites in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. In competition studies, the absolute affinities and slopes of the inhibition curves for several unlabelled ligands against [3H]bremazocine were not significantly different to those against [3H]U69593 and were consistent with binding to the kappa-opioid binding site. In the intestinal layers underlying the longitudinal muscle-myenteric plexus [3H]bremazocine, under kappa-selective conditions, recognized both a high and low affinity site. In contrast, [3H]U69593 bound to a homogeneous population of binding sites. The [3H]U69593 binding site and the [3H]bremazocine high affinity site demonstrated comparable characteristics to the single, kappa site identified in the longitudinal muscle layer. The nature of the low affinity site was not investigated due to difficulties associated with low specific binding, and its significance therefore remains to be investigated.     

The effects of the selective kappa-opioid agonist MR 2034 on the guinea-pig ileum

The effects of the selective kappa-opioid agonist MR 2034 on the guinea-pig ileum were compared with those produced by the mu-agonist morphine. MR 2034 induced acute tolerance and inhibited electrically evoked contractions to the same extent as morphine. The slope of the concentration-effect curve to MR 2034 was not significantly different from that of morphine. However, MR 2034 and morphine may act at their respective receptors since naloxone was 6.57 times more effective at inhibiting responses to morphine than MR 2034. MR 2034, like morphine, induced acute dependence as revealed by naloxone-induced contractions of tissues that had been incubated with MR 2034 for 5 h. The functional properties of kappa-opoid receptors are the same as mu-receptors in the guinea-pig ileum.     

Withdrawal contractures of guinea-pig isolated ileum after acute activation of kappa-opioid receptors.

1. The present study was undertaken to investigate firstly whether a brief exposure for 5 min of guinea-pig isolated ileum to the kappa-opioid agonist, U-50,488H produced a withdrawal contracture on addition of naloxone and secondly to ascertain whether the response was due to the activation of kappa-opioid receptors. 2. Naloxone (10(-6) M) did not elicit a response in preparations exposed to U-50,488H (5 x 10(-7) M-2 x 10(-6) M). However, after exposure to U-50,488H (5 x 10(-7) M), naloxone (10(-6) M) produced a strong contracture if the agonist was washed out 1 min before the addition of the antagonist. 3. The addition of naloxone (10(-6) M) to the ileum preparation exposed to U-50,488H (10(-7) M or lower) caused a response of similar intensity irrespective of whether the agonist had been washed out. 4. The selective kappa-opioid antagonist, nor-binaltorphimine (2.7 x 10(-9) M and 2.7 x 10(-7) M), injected before the opioid agonists, prevented the naloxone-induced contracture after exposure to U-50,488H (8 x 10(-8) M) but did not affect the contracture after exposure to morphine (5 x 10(-7) M). 5. Nor-binaltorphimine (2.7 x 10(-9) M) caused a contraction of the ileum preparation when injected 5 min after exposure to U-50,488H (8 x 10(-8) M) but not after morphine (5 x 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of the heterogeneity of the kappa-opioid receptors in guinea-pig cerebellum using selective and nonselective drugs

In guinea-pig cerebellum, saturation studies reveal that the nonselective opioid [3H]ethylketazocine has a binding capacity (R) of 6.79 pmol/g tissue which is similar to the sum of the individual R values of the mu-, delta- and kappa 1-selective opioids. Conversely, the binding parameters of the nonselective opioid [3H]bremazocine are best-fitted to a two-site model (Kd1 = 0.12 nM, R1 = 11.3 pmol/g tissue; Kd2 = 6.03 nM, R2 = 9.09 pmol/g tissue) with an R TOTAL value of 20.3 pmol/g tissue which is statistically different from the R value of [3H]ethylketazocine or the sum of R mu + R delta + R kappa 1. This suggests that [3H]bremazocine labels additional opioid binding sites. After suppression of the mu-, delta- and kappa 1-receptors, [3H]bremazocine binding is then best-fitted to a one-site model with a Kd value of 1.48 nM and an R value of 11.2 pmol/g tissue. Competition studies done against the binding of [3H]U69593 indicate that the opioid receptors labelled with this ligand are related to the kappa 1-receptor subtype. However, competition studies performed against the binding of [3H]bremazocine (under suppressed conditions) display a pharmacological profile related to another subtype of kappa-receptors previously described in guinea-pig brain as the kappa 2-receptors.     

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible antagonism of histamine H2 receptors in guinea-pig myocardium

Dissociation constants and receptor reserves for histamine acting at H2 receptors in guinea-pig right atria and papillary muscles were measured using the irreversible H2 receptor antagonist L-643,441. Histamine alone was less potent in papillary muscles than in right atria, and maximum responses of papillary muscles to histamine were more susceptible to depression by L-643,441 than were maximum responses of right atria. However, dissociation constants (-log KA values) for histamine calculated using 3 different doses of L-643,441 were 0.5 log unit greater in papillary muscles than in right atria. A more marked difference was found in the estimates of receptor reserve for histamine, which appeared to be much greater in right atria than papillary muscles.     

Release-modulating acetylcholine receptors in cholinergic neurones of the guinea-pig ileum.

1 Twitch responses of the guinea-pig ileum to electrical transmural stimulation (0.2 Hz) were smaller after a dose of acetylcholine (ACh) than before it. The magnitude of the post-ACh inhibition of twitch was dose-dependent. 2 The post-ACh inhibition of twitch could not be explained in terms of post-junctional desensitization and was not modified by guanethidine, thymoxamine, propranolol or naloxone. Inhibition of twitch also followed high frequency stimulation (10 Hz) but this inhibition, unlike that following ACh, was partially antagonized by naloxone. 3 Hexamethonium (C6) in concentrations known to block contractions to nicotine, potentiated the post-ACh inhibition of twitch and modified the pattern of recovery. An initial rapid phase followed by a slower phase was converted by C6 to an initial slow phase followed by a more rapid rate of recovery. 4 The C6-sensitive (nicotinic) component of twitch recovery after ACh was also dose-dependent and contributed greatly to the rapid recovery during the first minute after ACh washout, whereas during the same period the C6-resistant inhibition remained relatively constant; thereafter both components declined. The C6-resistant inhibition was considered to be due to the activation of prejunctional muscarinic receptors. 5 5-Hydroxytryptamine (5-HT) and nicotine also caused inhibition of twitch but the pattern of response differed from those due to ACh, the maximum inhibition usually being produced 1 min after recommencing stimulation. High doses of 5-HT produced inhibitory responses similar to those following ACh, whereas nicotine produced a characteristic triphasic pattern of response. 6 It is concluded that ACh acts on at least two prejunctional receptors subserving a modulatory role on transmitter release, a nicotinic receptor whose activation enhances ACh output and a muscarinic receptor whose activation leads to an inhibition of transmitter secretion.     

Antagonist discrimination between subtypes of tachykinin receptors in the guinea-pig ileum

The effects of substance P and eledoisin on spontaneous and electrically-evoked release of [3H]acetylcholine, and on smooth muscle were studied in the guinea-pig myenteric plexus-longitudinal muscle preparation preloaded with [3H]choline. Substance P and eledoisin caused transient increases in spontaneous release of [3H]-acetylcholine and in longitudinal muscle tone. Both tachykinins were equipotent in contracting the muscle, but eledoisin was more potent than substance P in eliciting [3H]acetylcholine release. The release caused by substance P was enhanced in the presence of naloxone and scopolamine which suggests that the release is modulated through opioid and muscarinic receptors. Substance P and eledoisin inhibited the release of [3H]acetylcholine evoked by electrical stimulation at 0.1 Hz. The inhibition was not due to an activation of alpha-adrenoceptors, histamine or opioid receptors. The substance P antagonists (D-Pro2, D-Trp7,9)SP (10 and 30 microM) and (Arg5, D-Trp7,9, Nle11)SP5-11 (1 and 10 microM) competitively antagonized both the contractile effects of substance P and eledoisin, and the inhibition by the tachykinins of the electrically-evoked release of [3H]acetylcholine. The increase in spontaneous [3H]acetylcholine release elicited by substance P and eledoisin was not prevented by the substance P antagonists. The results suggest that the neuronal receptor whose activation causes inhibition of acetylcholine release and the smooth muscle receptor correspond to the SP-P type, whereas the neuronal receptor mediating an increase in spontaneous acetylcholine release is of the SP-E type. The two antagonists, (D-Pro2, D-Trp7,9)SP and (Arg5, D-Trp7,9, Nle11)SP5-11, selectively block only the SP-P receptor.     

Effect of selective phosphodiesterase inhibitors on synaptic transmission in the guinea-pig ileum

The effect of selective phosphodiesterase (PDE) inhibitors was studied on neural transmission within the enteric nervous system employing a two-compartment bath (containing the oral and the anal end of a segment of guinea-pig ileum, respectively). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 45 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded. The partitioned bath enables PDE inhibitors and other drugs to be applied to enteric nerve pathways (in the anal compartment) without interfering with the recording of the smooth muscle contraction in the oral compartment. The PDE 4 inhibitors rolipram (0.01–10 μM) and Ro-20-1724 (0.01–10 μM) significantly (P<0.01) inhibited (10–91% and 9–83%, respectively) the nerve-mediated contractions. When both rolipram and Ro-20-1724 were tested after phentolamine (1 μM) or yohimbine (0.1 μM), they were significantly (P<0.01) less effective. By contrast prazosin (1 μM) was ineffective. Vinpocetine (50 μM), milrinone (30 μM) and zaprinast (100 μM), which inhibit PDE 1, 3 and 5, respectively, did not modify the nerve-mediated contractions. 8-Bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cyclic AMP) or N⁶,2’-O-dibutyryladenosine 3’,5’ cyclic monophosphate (dibutyryl cyclic AMP), two analogues of cyclic AMP, at lower concentrations (0.1–1 μM) significantly (P<0.01) inhibited (15–73% and 5–49%, respectively) the nerve-mediated contractions, while at higher concentrations (10–100 μM) they caused a significant (P<0.01) potentiating (48–68% and 77–78%, respectively) effect. These results indicate that inhibition of PDE 4 (but not PDE 1, PDE 3 or PDE 5) produces a depression of neural transmission within the enteric nervous system, possibly by releasing noradrenaline acting at α₂-adrenoceptors on enteric neurons. Received: 21 November 1997 / Accepted: 11 March 1998     

Pyrrolizidine alkaloids: Actions on muscarinic receptors in the guinea-pig ileum

1. Eleven pyrrolizidine alkaloids have been tested on the isolated guinea-pig ileum preparation.2. Platyphylline, supinine, heleurine and cynaustraline were more potent in antagonizing responses to acetylcholine and carbachol than responses to histamine. Their anticholinergic activity appeared to involve a competitive mechanism.3. Lasiocarpine, monocrotaline, spectabiline, sarracine, 7-angelylheliotridine, heliotrine and senecionine had similar antagonistic potencies against responses to both acetylcholine and histamine.4. The alkaloids had no appreciable activity as antagonists of acetylcholine in the isolated toad rectus abdominis preparation.5. These results are discussed with respect to interactions of the alkaloids at receptor sites involved in anticholinergic activity at the muscarinic receptor.     

Characterization of receptors on postganglionic cholinergic neurons in the guinea-pig isolated ileum

Dopamine, apomorphine, noradrenaline and isoprenaline reduced the response of the isolated guinea-pig ileum to exogenous acetylcholine by a maximum of 40%. Propranolol reversed this inhibition whilst phentolamine and pimozide were ineffective, suggesting that the drugs were acting on a post-synaptic β-adrenoceptor. The same agonists were more effective as inhibitors of the response to transmural electrical stimulation of the ileum, lower doses producing almost complete inhibition. This inhibition was partially antagonized by phentolamine, pimozide and propranolol. Clonidine proved to be the most potent inhibitor of the response to transmural electrical stimulation, whilst phenylephrine was ineffective. pA₂ determinations showed that phentolamine was a potent antagonist of clonidine but a weak antagonist of apomorphine whilst for pimozide the opposite was true. The results suggest that there are two populations of prejunctional receptors on the cholinergic nerves innervating the smooth muscle of the guinea-pig ileum. One receptor is similar to a classical prejunctional α-adrenoceptor and the other resembles a central dopamine receptor.     

Characterization of receptors on postganglionic cholinergic neurons in the guinea-pig isolated ileum.

Dopamine, apomorphine, noradrenaline and isoprenaline reduced the response of the isolated guinea-pig ileum to exogenous acetylcholine by a maximum of 40%. Propranolol reversed this inhibition whilst phentolamine and pimozide were ineffective, suggesting that the drugs were acting on a post-synaptic beta-adrenoceptor. The same agonists were more effective as inhibitors of the response to transmural electrical stimulation of the ileum, lower doses producing almost complete inhibition. This inhibition was partially antagonized by phentolamine, pimozide and propranolol. Clonidine proved to be the most potent inhibitor of the response to transmural electrical stimulation, whilst phenylephrine was ineffective. pA2 determinations showed that phentolamine was a potent antagonist of clonidine but a weak antagonist of apomorphine whilst for pimozide the opposite was true. The results suggest that there are two populations of prejunctional receptors on the cholinergic nerves innervating the smooth muscle of the guinea-pig ileum. One receptor is similar to a classical prejunctional alpha-adrenoceptor and the other resembles a central dopamine receptor.     

Antagonism by imidazoline-type drugs of muscarinic and other receptors in the guinea-pig ileum

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10(-5) m. A dissociation constant (KB) of 3.6 microm for the antagonist was calculated. At higher concentrations, 3 x 10(-5) and 10(-4) m, of the antagonist, the agonist dose-response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10(-4) m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10(-5) m produced two-, three- and four-fold shift of the carbachol dose-response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the alpha-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine.     

Effect of dithiothreitol on histamine receptors in rabbit colon and guinea-pig ileum

1. Dithiothreitol, an agent which reduces disulphide linkages to sulphydryl groups, potentiated the contractile responses of the rabbit colon and guinea-pig ileum to histamine, but had no effect on the resting tone or on the responses of these preparations to acetylcholine. 2. The potentiation was greater than that produced by antagonism of histamine's weak inhibitory action by metiamide, and still occurred after blockade of H2-receptors with metiamide; it is concluded that DTT potentiates responses to stimulation of H1-receptors in both preparations. 3. In this respect, these H1-receptors resemble those in vascular smooth muscle in the rabbit but not in the guinea-pig; it is concluded that there is a tissue variation rather than a species variation in the response of Hi-receptors in the rabbit and guinea-pig to DTT.     

Functional group I metabotropic glutamate receptors in submucous plexus of guinea-pig ileum

Intracellular recording methods and immunostaining revealed the existence of functional group I metabotropic glutamate receptors (mGluRs) in submucous plexus neurons of guinea-pig ileum. Selective group I, but not groups II or III metabotropic glutamate receptor agonists induced concentration-dependent, slowly-activating depolarizing responses. Group I metabotropic glutamate receptor antagonism observed with (S)-4-carboxyphenylglycine (S-4-CPG) (100 - 600 microM) was competitive as determined by Schild analysis (pA(2)=3.81+/-0.02). Neither the group II and III metabotropic nor ionotropic glutamate receptor antagonists altered responses evoked by group I receptor agonists. Immunoreactivities for metabotropic glutamate 1alpha and 5 receptors were found to locate exclusively in neurons in the submucous plexus of guinea-pig ileum with the highest density around the cell bodies. The results suggest that group I metabotropic glutamate receptors are functionally expressed in the submucous plexus of guinea-pig small intestine.     

Characterization of 5-hydroxytryptamine receptors mediating mucosal secretion in guinea-pig ileum.

1. The receptor subtypes through which 5-hydroxytryptamine (5-HT) increases electrolyte secretion across the mucosa of guinea-pig ileum were studied. 2. Flat sheep preparations of guinea-pig mucosa plus submucosa were placed in Ussing chambers and the short circuit current (ISC), an index of net electrogenic electrolyte transport across the mucosa, was measured under voltage clamp conditions. 3. Low concentrations of 5-HT (10-300 nM) evoked monophasic increases in ISC which were significantly reduced by hyoscine (100 nM), tetrodotoxin (TTX, 300 nM) and the 5-HT2 receptor antagonist, ketanserin (3-300 nM). 4. Higher concentrations of 5-HT (1-10 microM) produced biphasic responses which were reduced by hyoscine (100 nM), TTX (300 nM), ketanserin (3-300 nM) and also by the 5-HT3 receptor antagonists, granisetron (1 microM) and ICS 205-930 (100 nM). 5. 2-Methyl-5-HT (1-100 microM) and alpha-methyl-5-HT (30 nM-30 microM), agonists at 5-HT3 and 5-HT2 receptors respectively, also evoked ISC increases. These responses were reduced by hyoscine (100 nM) and abolished by TTX (300 nM) and the respective receptor antagonists, granisetron (1 microM) and ketanserin (30 nM). 6. The 5-HT4 receptor antagonist, SDZ 205-557 (300 nM) had no effect on the response to 5-HT. 7. The TTX-resistant response to 5-HT was not affected by 5-HT2,3 or 4 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bremazocine is an agonist at kappa-opioid receptors and an antagonist at mu-opioid receptors in the guinea-pig myenteric plexus.

The agonist and antagonist activity of bremazocine at opioid receptors in the guinea-pig myenteric plexus preparation was determined in untreated tissues and in tissues in which either mu-9 or kappa-opioid receptors were blocked preferentially. After pretreatment of the tissue with beta-funaltrexamine for 90 min followed by washing out, the IC50 value of the selective mu-ligand [D-Ala2,MePhe4,Gly-ol5]enkephalin was increased 67 fold whereas the IC50 values of the selective kappa-ligand U-69,593 and of the non-selective kappa-ligand bremazocine were not significantly changed. In this experimental design bremazocine acted only on kappa-receptors. After pretreatment of the tissue with beta-chlornaltrexamine and 10 microM of the mu-ligand for 30 min followed by washout, the IC50 value of the mu-ligand was increased 2 fold whereas the IC50 value of the selective kappa-ligand was increased 32 fold and that of bremazocine 62 fold. Under these experimental conditions, it was shown that bremazocine is an antagonist against [D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-receptor (Ke = 1.6 nM). The residual agonist activity of bremazocine is at the kappa-receptor. In naive myenteric plexus preparations the mu-antagonist activity of bremazocine cannot be demonstrated because its potency at the kappa-receptor is very high. This dual action may be of importance for the responses of bremazocine in other peripheral and central tissues.     

Similarity between mu-opioid receptors in mouse vas deferens and guinea-pig ileum.

The effects of the opioid receptor agonist RX783006 and of the opioid receptor partial agonist (+)-meptazinol have been examined on electrically-induced twitch responses of the guinea-pig isolated ileum and of the mouse isolated vas deferens. Log10 concentration-tissue state curves were determined for (+)-meptazinol and for RX783006, alone, in combination and, when appropriate, in the presence of naloxone (30 nM). Analysis of these log10 concentration-tissue state curves using the null equations derived and verified in the previous paper allows quantitation of the characteristics of the interaction of (+)-meptazinol with the opioid receptors in these tissues. The results indicate that the apparent differences in the actions of (+)-meptazinol on isolated electrically-stimulated guinea-pig ileum and mouse vas deferens can be accounted for without the need to postulate differences between mu-opioid receptors in these two tissues.     

Influence of temperature on the effects of mu-, delta- and kappa-opioid receptor agonists in the guinea-pig ileum myenteric plexus.

Electrically stimulated guinea-pig ileum myenteric plexus-longitudinal muscle was used to determine if changes in temperature alter the inhibitory effects of DAGO ([D-Ala2,N-MePhe4,Gly5-ol]enkephalin, mu-agonist), DPDPE ([D-Pen2,-Pen5] enkephalin, delta-agonist) and U-50,488H (trans-3,4-dichloro)-N-methyl-N-[2-(1- pyrrolidynyl)cyclohexyl]benzeneacetamide methane sulfonate, kappa-agonist). The potency (expressed as the concentration which produces 50% inhibition, IC50) of DAGO and DPDPE was significantly (P < 0.05) decreased at 30 degrees C (8.8 +/- 2.7 x 10(-9) and 8325.2 +/- 1070 x 10(-9) M), when compared to the potency at 37 degrees C (3.8 +/- 0.3 x 10(-9) and 6298.6 +/- 320 x 10(-9) M). Higher temperature (40 degrees C) did not modify the potency of DAGO or DPDPE compared to that at 37 degrees C. However, the potency of U-50,488H was significantly (P < 0.01) increased at 40 degrees C (0.7 +/- 0.0 x 10(-9) M) versus 37 degrees C (2.4 +/- 0.9 x 10(-9) M) or 30 degrees C (2.5 +/- 0.3 x 10(-9) M). The kappa-agonist was more potent than DAGO or DPDPE at 30 or 40 degrees C. These data demonstrate that changes in temperature can alter the potency of opioid agonists.