Hyoscine-resistant peristalsis in guinea-pig ileum

The effect of hyoscine on the peristaltic activity of the proximal and distal ileum of the guinea-pig was studied. Hyoscine strongly impaired peristalsis as indicated by the elevation of the threshold pressure and by the increased number of incomplete peristalses and blockades. Functional activity of the circular musculature was more markedly impaired. However, particularly in the distal ileum, complete peristalses occurred even after 70 min exposure to hyoscine at a concentration of 10^?6 g/ml. A tenfold increase in hyoscine concentration failed to producedfurther impairment of peristaltic activity and of the oral reflex contraction. The activity which remained in the presence of hyoscine was blocked by methysergide and by d-tubocurarine. The hypothesis is advanced that once the muscarinic receptors have been blocked, increased radial stretch of the circular coat results in activation of a separate, tetrodotoxin sensitive, excitatory nervous pathway, which is sufficient to maintain a discrete degree of peristaltic activity.     

CB1 cannabinoid receptor ligands

The CB1 receptor is expressed in the central nervous system and numerous other tissues including heart, lung and uterus and has been recognized as an important therapeutic target for pain, appetite modulation, glaucoma, multiple sclerosis and other indications. An interesting feature of this GPCR is its ability to be activated by a number of structurally different classes of compounds, thus, raising the possibility of multiple activated forms of the receptor. Understanding of the structure-activity relationships of cannabinergic ligands has paved the road for the development of novel ligands exhibiting receptor subtype selectivity and efficacy. This review highlights the important CB1 cannabinergic ligands developed to date.     

Modulation of peristalsis by NK3 receptor antagonism in guinea-pig isolated ileum is revealed as intraluminal pressure is raised

1. NK(3) tachykinin receptors mediate slow excitatory transmission in the enteric nervous system and play a role in reflexes induced by the intestinal stretch or mucosal compression. However, there is little evidence to suggest that these receptors are important in peristalsis. We have examined the effects of the NK(3) receptor antagonist, talnetant, on peristalsis in guinea-pig isolated ileum induced by optimal and by supra-maximal distension pressures. 2. At the guinea-pig NK(3) receptor, talnetant was shown to have high affinity (pK(B) 8.8) and selectivity over the guinea-pig NK(1) and NK(2) receptors. 3. Peristaltic waves in the ileum elicited by optimal distension pressures (1-3 cmH(2)O) were unaffected by talnetant at a supra-maximal concentration (250 nm). 4. Distension at a higher pressure (4 cmH(2)O) induced peristalsis in which there was incomplete closure of the lumen during each peristaltic wave and an increase in the periods of inactivity observed between bursts of peristaltic activity. The addition of talnetant (250 nm) increased the number of peristaltic events by reducing these periods of inactivity and thus, increased the productivity of the peristaltic reflex. 5. The data suggest that NK(3) receptors are not involved in the modulation of peristaltic movements by physiological stimuli, but they may have a role in modulation of reflexes in extreme or pathological conditions.     

Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids.

1. A recording method was developed to measure physiological parameters of the preparatory and emptying phases of peristalsis in vitro. This method enabled measurement of: the compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension); longitudinal muscle contraction during the preparatory phase; the threshold volume required to trigger the emptying phase; the maximal ejection pressure and the average power generated during the emptying phase, which reflects the rate at which the intestine performs work. Modulation of these parameters by exogenous and endogenous opioids acting at mu, kappa and delta opioid receptors was investigated. 2. The compliance of the intestinal wall during the preparatory phase was reduced by the mu opioid receptor agonist, [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin (DAMGO) but not by the kappa agonist, dynorphin, or the delta agonist, [D-penicillamine2, D-penicillamine5] enkephalin (DPDPE). Reflex contraction of the longitudinal muscle during the preparatory phase was inhibited by DAMGO, dynorphin and DPDPE. The threshold volume required to trigger the emptying phase of peristalsis was increased by DAMGO, dynorphin and DPDPE. 3. The maximal ejection pressure generated during the emptying phase was reduced by dynorphin and DPDPE, but not by DAMGO. The average power generated by the intestine when emptying was not altered by any of the agonists. 4. Electrically stimulated contractions of longitudinal muscle in strips of longitudinal muscle-myenteric plexus were not inhibited by DPDPE. Similarly, DPDPE did not significantly inhibit electrically induced contraction of circular muscle in strips of circular muscle-myenteric plexus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of SKF 10,047 (N-allyl-normetazocine) on peristalsis and longitudinal muscle contractions of the isolated guinea-pig ileum

Summary The purpose of this study was to investigate the differential involvement of distinct types of opioid receptors in the modulation of intestinal peristalsis compared to electrically induced longitudinal muscle contractions.Like naloxone, the proposed -agonist and -antagonist SKF 10,047 (N-allyl-normetazocine) dose-dependently enhanced peristaltic circular muscle contractions in the isolated guinea-pig ileum. Pre-application of SKF 10,047 at a concentration which itself enhanced peristalsis by 20% on average strongly attenuated the inhibition of peristalsis produced by opioids previously proposed to act via -opioid-receptors in the guinea-pig ileum, i.e. normorphine, -endorphin, d-Ala²-d-Leu⁵-enkephalin and d-Ser²-l-Leu⁵-enkephalyl-Thr, but less strongly attenuated the inhibition produced by compounds suggested to act via -opioid-receptors in this tissue, i.e. ethylketazocine and dynorphin (1–13). In contrast to its effect on peristalsis, SKF 10,047 inhibited the electrically induced contractions of the myenteric plexus-longitudinal muscle preparation in a naloxonereversible fashion.It may be concluded that - and -opioid receptors are of a greater functional significance than -receptors in the control of peristalsis. -Receptors might participate predominantly in modulating the release of acetylcholine which underlies the electrically induced longitudinal muscle contraction.     

Evidence for both inverse agonism at the cannabinoid CB1 receptor and the lack of an endogenous cannabinoid tone in the rat and guinea-pig isolated ileum myenteric plexus-longitudinal muscle preparation

Background and purpose:

Cannabinoid receptor agonists reduce intestinal propulsion in rodents through the CB₁ receptor. In addition to its antagonistic activity at this receptor, rimonabant (N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxyamide) alone augments intestinal transit. Using rat and guinea-pig ileum MPLM (myenteric plexus-longitudinal muscle) preparations, we investigated whether the latter effect was through inverse agonism or antagonism of endocannabinoid agonist(s).

Experimental approach:

Inverse agonism was investigated by comparing the maximal enhancement of electrically evoked contractions of the MPLM by two CB₁ receptor antagonists, AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and O-2050 [(6aR,10aR)-3-(1-methanesulphonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6-H-dibenzo[b,d]pyran], with that produced by rimonabant. To reveal ongoing endocannabinoid activity, effects of inhibiting endocannabinoid hydrolysis by fatty acid amide hydrolase (FAAH) using AA-5HT (arachidonyl-5-hydroxytryptamine), PMSF (phenylmethylsulphonyl fluoride) or URB-597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate), or putative uptake using VDM-11 [(5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] was evaluated.

Key results:

The presence of CB₁ receptors was revealed by antagonism of exogenous anandamide, arachidonylethanolamide (AEA) and WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] by rimonabant. The rank order of potentiation of contractions was AM 251 > rimonabant > O-2050. Neither the FAAH inhibitors nor VDM-11 affected electrically evoked contractions. Each FAAH inhibitor increased the potency of AEA but not WIN 55,212-2. VDM-11 did not alter the inhibitory effect of AEA.

Conclusions and implications:

The different levels of maximal potentiation of contractions by the CB₁ receptor antagonists suggest inverse agonism. The potentiation of the action of AEA by the FAAH inhibitors showed that FAAH was present. The lack of effect of FAAH inhibitors and VDM-11 alone on electrically evoked contractions, and on the potency of exogenous AEA suggests that pharmacologically active endocannabinoids were not released and the endocannabinoid transporter was absent. Thus, the CB₁ receptor antagonists behave as inverse agonists.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Inhibition of peristalsis in guinea-pig isolated ileum and colon by drugs that block prostaglandin synthesis.

1 Methods of analysing peristaltic activity have been evaluated by the use of recordings of longitudinal and circular muscle activity and of propulsion in whole segments of guinea-pig ileum and colon. 2 Some prostaglandin synthesis inhibitors, and antagonists of prostaglandin action were tested for their suitability for studying the role of prostaglandins in peristalsis. Aspirin was suitable; at 10-200 mug/ml it had little effect on responses of longitudinal muscle strips of the guinea-pig ileum to acetylcholine (ACh), histamine, nicotine or prostaglandin E2. Indomethacin (1-4 mug/ml) reduced responses to nicotine and prostaglandin E2. The prostaglandin antagonists polyphloretin phosphate and SC-19220 reduced contractions of ileal longitudinal muscle caused by nerve excitation with either nicotine or transmural stimulation. 3 Aspirin (20-100 mug/ml) or indomethacin (1-4 mug/ml) applied serosally greatly inhibited all aspects of peristalsis in guinea-pig ileum and colon. Inhibition of peristalsis of the ileum by aspirin was antagonized by prostaglandin E2 and that by indomethacin was removed by prostaglandin F2alpha or ACh. Inhibition of colonic peristalsis by aspirin was antagonized by prostaglandin E2 but rarely by ACh, and that by indomethacin by prostaglandin E1 or E2. Mucosal application of aspirin had little effect on either ileum or colon but indomethacin caused some inhibition. 4 These results support the supposition that prostaglandins contribute to peristaltic activity.     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS).
2. The cannabinoid receptor agonist WIN 55,212-2 (1–1000 nM) and the putative endogenous ligand anandamide (0.1–100 μM) both produced a concentration-dependent inhibition of the cholinergic (9–57% and 1–51% inhibition) and NANC (9–55% and 2–57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P.
3. Apamin (30 nM), a blocker of Ca²⁺-activated K⁺ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. N^G-nitro-L-arginine methyl ester (100 μM), an inhibitor of nitric oxide synthase, or naloxone (1 μM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions.
4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB₁ receptor antagonist SR 141716A (10–1000 nM).
5. In absence of other drugs, SR 141716A (1–1000 nM) enhanced cholinergic (1–45% increase) and NANC (2–38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P.
6. It is concluded that activation of prejunctional cannabinoid CB₁ receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K⁺ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.

     

Effect of metoclopramide on peristalsis and antiperistalsis in isolated frog stomach and guinea-pig ileum

The action of metoclopramide was studied on peristaltic as well as antiperistaltic activity in frog stomach and guinea-pig ileum. It was observed that metoclopramide enhances normal peristaltic activity by acting on dopamine-receptors whereas it inhibits antiperistalsis by some unknown mechanism. It is, therefore, inferred that inhibition of antiperistaltic activity might be an additional mechanism for its antiemetic effect. It also explains the pharmacological basis for its use in oesophagitis due to gastro-oesophageal reflux.     

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum

1. The effect of cannabinoid receptor agonists was studied in guinea-pig myenteric neurones in vitro by use of conventional intracellular recording techniques.
2. Exposure of myenteric neurones of the S-cell type to the cannabinoid receptor agonists WIN 55,212-2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively.
3. The depressant effect of WIN 55,212-2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude-time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect.
4. The inhibitory effect of WIN 55,212-2 appeared to be concentration-dependent over the range 1–100 nM. WIN 55,212-3, its (−)-enantiomer (100 nM), was inactive.
5. The cannabinoid CB₁ receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212-2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)-induced depolarizations in 42% of neurones tested (5/12).
6. When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9).
7. WIN 55,212-2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM).
8. It is concluded that cannabinoid-induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB₁ receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S-neurones and their synapsing cholinergic and non-cholinergic, non-adrenergic terminals are not endowed with cannabinoid receptors.

     

The effect of PGE1 on peristalsis and on perivascular nerve inhibition of peristaltic activity in guinea-pig isolated ileum

The effect of PGE₁ on peristalsis of guinea-pig isolated ileum was examined using a modified Trendelenburg method to evoke and record peristaltic activity. PGE₁ (14 nm , 0·11 μm and 0·56 μm ) increased peristaltic activity of both longitudinal and circular muscle, mainly by increasing the amplitude of contraction. Preparations of ileum subjected to a ‘minimal’ peristaltic stimulus were more sensitive to the effects of PGE, than were preparations subjected to a ‘just-maximal’ peristaltic stimulus. The inhibition of peristaltic activity caused by perivascular nerve stimulation was antagonized by 0·56 μm PGE₁ but slightly increased by 14 nm PGE₁.     

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or co-incubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC₅₀ 73 nM), but had no additive effect with atropine or TTX. The cannabinoid CB₁ receptor antagonist SR 141716 (pA₂ 8.2), but not the CB₂ receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction.  These results provide functional evidence of the existence of prejunctional cannabinoid CB₁-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB₁ receptors naturally expressed in the human gut.     

Agonist selective regulation of G proteins by cannabinoid CB(1) and CB(2) receptors

We have examined the ligand regulation and G protein selectivity of the human cannabinoid CB(1) and CB(2) receptors by an in situ reconstitution technique directly measuring G protein activation. Membranes from Spodoptera frugiperda cells expressing CB(1) and CB(2) receptors were chaotrope extracted to denature endogenous GTP-binding proteins. The ability of the receptors to catalyze the GDP-GTP exchange of each G protein was then examined with purified bovine brain G(i) and G(o). Activation of CB(1) receptors produced a high-affinity saturable interaction for both G(i) and G(o). Agonist stimulation of CB(2) receptors also resulted in a high-affinity saturable interaction with G(i). In contrast, CB(2) receptors did not interact efficiently with G(o). G protein activation was then examined with a diverse group of ligands. For the interaction of CB(2) receptors with G(i), HU210 was the only compound tested that demonstrated maximal activation. In contrast, WIN55,212 (64%), anandamide (42%), and Delta(9)-tetrahydrocannabinol (Delta(9)-THC) (44%) all initiated submaximal levels of G protein activation. For CB(1) receptor-catalyzed activation of G(i), HU210, WIN55,212, and anandamide all elicited maximal activation, whereas Delta(9)-THC (56 +/- 6%) caused only partial G(i) activation. In contrast, only HU210 effected maximal CB(1) stimulation of G(o), with anandamide, WIN55, 212, and Delta(9)-THC all stimulating between 60 and 75% compared with HU210. These data demonstrate that different agonists induce different conformations of the CB(1) receptor, which in turn can distinguish between different G proteins. Our data thus demonstrate agonist-selective G protein signaling by the CB(1) receptor and suggest that therapeutic agents may be designed to regulate individual G protein-signaling pathways selectively.     

5-HT7 receptors mediate the inhibitory effect of 5-HT on peristalsis in the isolated guinea-pig ileum

The study was undertaken to investigate the 5-HT receptor mediating the inhibitory effect of 5-HT on peristalsis in the guinea-pig isolated ileum. The facilitatory and inhibitory effects were measured as the decrease and increase, respectively, in the intraluminal pressure required to trigger peristalsis. In the presence of 5-HT(2/3&4) receptor antagonists ketanserin (0.1 micro M), granisetron (1 micro M) and SB-204070 (1 micro M), a cumulative addition (0.1-100 micro M) of 5-HT or 5-carboxamidotryptamine, but not 2-methyl-5-HT produced a concentration-dependent increase in the threshold required to trigger peristalsis. The 5-HT(7) receptor selective antagonist SB-269970-A (0.01-1 micro M) or methiothepin (0.01-0.1 micro M) concentration-dependently antagonised this response to 5-HT. SB-269970-A (1 micro M) and methiothepin (1 micro M) were also able to restore peristalsis in tissues in which peristalsis was inhibited by a prior addition of 30 micro M of 5-HT. The results indicate an involvement of 5-HT(7) receptors in the inhibitory effect of 5-HT on peristalsis in the guinea-pig ileum.     

The effect of PGE1 on peristalsis and on perivascular nerve inhibition of peristaltic activity in guinea-pig isolated ileum.

The effect of PGE1 on peristalsis of guinea-pig isolated ileum was examined using a modified Trendelenburg method to evoke and record peristaltic activity. PGE1 (14 nM, 0.11 microM and 0.56 microM) increased peristaltic activity of both longitudinal and circular muscle, mainly by increasing the amplitude of contraction. Preparations of ileum subjected to a 'minimal' peristaltic stimulus were more sensitive to the effects of PGE1 than were preparations subjected to a 'just-maximal' peristaltic stimulus. The inhibition of peristaltic activity caused by perivascular nerve stimulation was antagonized by 0.56 microM PGE1 but slightly increased by 14 nM PGE1.     

Modulation of gastric emptying and gastrointestinal transit in rats through intestinal cannabinoid CB(1) receptors

We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((-)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB(1)-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID(50) range 1.0-3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB(1) receptor mechanisms in gastrointestinal transit delay by specific agonists.     

Recent advances in the development of selective ligands for the cannabinoid CB(2) receptor

Two subtypes of the mammalian cannabinoid receptor have been identified and successfully cloned since 1990. The CB(1) receptor is primarily located in the central nervous system and the CB(2) receptor is almost exclusively expressed in cells of the immune system. The CB(1) and CB(2) receptors are both G-protein coupled receptors and are involved in the inhibition of adenylate cyclase. The CB(2) receptor is of particular importance due to its involvement in signal transduction in the immune system, making it a potential target for therapeutic immune intervention. A number of these selective ligands are derivatives of traditional dibenzopyran based cannabinoids. These include the very recently synthesized (2'R)-1-methoxy-3-(2'-methylbutyl)- Delta (8)-THC (JWH-359) which has a 224 fold selectivity for the CB(2) receptor, readily comparable to the well known 1-deoxy-3-(1',1'-dimethylbutyl)- Delta (8)-THC (JWH-133) which has 200 fold selectivity for the CB(2) receptor. Several 9-hydroxyhexahydrocannabinols have also been synthesized and are found to be selective high affinity ligands for the CB(2) receptor. These are 1-deoxy-9beta-hydroxy-dimethylhexylhexahydrocannabinol (JWH-361, K(i) = 2.7 nM) and 1-deoxy-9beta-hydroxy-dimethylpentylhexahydrocannabinol (JWH-300, K(i) = 5.3 nM). CB(2) selective cannabi-mimetic indoles include 1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpholine]ethyl)-5-methoxyindole (L768242), (R)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) and 1-propyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), which exhibit significant selectivity for the CB(2) receptor coupled with weak affinity for the CB(1) receptor. Bristol-Meyer Squibb has produced a phenylalanine derived cannabimimetic indole which possesses high CB(2) affinity (K(i) = 8 nM) and very low affinity for the CB(1) receptor (K(i) = 4000 nM). This review will discuss the current advances and recent results in the structure-activity relationships (SAR) of selective ligands for the cannabinoid CB(2) receptor.     

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.     

Chemically distinct ligands promote differential CB1 cannabinoid receptor-Gi protein interactions

To understand how structurally distinct ligands regulate CB(1) receptor interactions with Gi1, Gi2, and Gi3, we quantified the Galphai and betagamma proteins that coimmunoprecipitate with the CB(1) receptor from a detergent extract of N18TG2 membranes in the presence of ligands. A mixture of A, R, G(GDP) (or G_), and ARG(GDP) (or ARG_) complexes was observed in the presence of aminoalkylindole (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2) for all three RGalphai complexes, cannabinoid desacetyllevonantradol for Galphai1 and Galphai2, and eicosanoid (R)-methanandamide for Galphai3. Desacetyllevonantradol maintained RGalphai3 complexes and (R)-methanandamide maintained RGalphai1 and RGalphai2 complexes even in the presence of a nonhydrolyzable GTP analog. The biaryl pyrazole antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716) maintained all three RGalphai complexes. Gbeta proteins, and to a certain extent Ggamma2, exhibited the same association/dissociation pattern as the Galpha proteins. A GDP analog had no influence on any of these association/dissociation reactions and failed to promote sequestration of G proteins. These results can be explained by invoking the existence of an inverse agonist-supported inactive state in the ternary complex equilibrium model. WIN 55,212-2 behaves as an agonist for all three Gi subtypes; SR141716 behaves as an inverse agonist for all three Gi subtypes; desacetyllevonantradol behaves as an agonist for Gi1 and Gi2, and an inverse agonist at Gi3; and (R)-methanandamide behaves as an inverse agonist at Gi1 and Gi2, and an agonist at Gi3. These ligand-selective G protein responses imply that multiple conformations of the receptor could be evoked by ligands to regulate individual G proteins.