Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.

Contact heat thermal threshold testing in beagle dogs: baseline reproducibility and the effect of acepromazine, levomethadone and fenpipramide.

BACKGROUND: In this methodology article a thermal threshold testing device designed to test nociception in cats was assessed in six dogs. The purpose of this study was to investigate baseline reproducibility of thermal thresholds obtained by the contact heat testing device, to assess the influence of acepromazine and levomethadone and fenpipramide in dogs. The relationship between change in nociceptive thermal threshold and the opioid's plasma concentration was determined. Six adult beagle dogs received levomethadone (0.2 mg/kg), acepromazine (0.02 mg/kg) or saline placebo by intramuscular injection (IM) in a randomized cross-over design. Three baseline nociceptive thermal threshold readings were taken at 15 minutes intervals prior to treatment. Further readings were made at 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 420 and 480 minutes after injection. A sedation score was assigned at every reading. Four saline placebo treatments were performed to assess baseline reproducibility. Levomethadone serum concentrations were measured prior and 0.5, 1, 2, 4, 8, 12 and 24 hours after drug dosing in a separate occasion. RESULTS: Acepromazine did not seem to increase the thermal threshold at any time. After levomethadone there was a significant rise of the thermal threshold between 15 to 120 minutes at serum concentrations between 22.6-46.3 ng/mL. Baseline reproducibility was stable in adult beagle dogs. CONCLUSION: The thermal threshold testing system is a suitable device for nociceptive threshold testing in dogs.

Cardiovascular effects of a proprietary l-methadone/fenpipramide combination (Polamivet) alone and in addition to acepromazine in healthy Beagle dogs.

OBJECTIVE: To determine the cardiovascular effects of a proprietary l-methadone/fenpipramide combination (Polamivet) alone and in addition to acepromazine in dogs. STUDY DESIGN: Prospective, randomized, experimental crossover study. ANIMALS: Five adult healthy Beagle dogs (one male and four females, weighing 12.8-16.4 kg). METHODS: Dogs were instrumented for haemodynamic measurements whilst anaesthetized with isoflurane. Three hours after recovery dogs received 0.025 mg kg(-1) acepromazine (AP) or saline (SP) IM followed by 0.5 mg kg(-1) L-methadone/ 0.025 mg kg(-1) fenpipramide IV after 30 minutes. Cardiac output using thermodilution, heart rate, mean arterial pressure (MAP), central venous pressure (CVP), mean pulmonary artery pressure (MPAP), pulmonary artery occlusion pressure (PAOP), haemoglobin concentration, arterial and mixed-venous blood gas analysis were measured and sedation evaluated at baseline (BL), 30 minutes after acepromazine or saline IM (A/S), 5 minutes after L-methadone/fenpipramide IV application (35), every 15 minutes for 1 hour (50, 65, 80, 95 minutes) and every hour until baseline cardiac output was regained. Standard cardiovascular parameters were calculated. Data were analyzed by repeated measures anova and paired t-tests with p < 0.05 considered significant. RESULTS: Baseline measurements did not differ. Cardiac index decreased after acepromazine administration in treatment AP (p = 0.027), but was not significantly influenced after l-methadone/fenpipramide injection in either treatment. In both treatments heart rate did not change significantly over time. Stroke volume index increased after A/S in both treatments (p = 0.049). Systemic vascular resistance index, MAP, CVP, MPAP, and pulmonary vascular resistance index did not change significantly after either treatment and did not differ between treatments. Dogs were deeply sedated in both treatments with a longer duration in treatment AP. CONCLUSIONS AND CLINICAL RELEVANCE: In healthy dogs the dose of l-methadone/fenpipramide used in this study alone and in combination with acepromazine induced deep sedation without significant cardiovascular changes.

Diphenylmethylene hydroxamic acids as selective class IIa histone deacetylase inhibitors.

We have identified a series of diphenylmethylene hydroxamic acids as novel and selective HDAC class IIa inhibitors. The original hit, N-hydroxy-2,2-diphenylacetamide (6), has sub-micromolar class IIa HDAC inhibitory activity, while the rigidified oxygen analogue, N-hydroxy-9H-xanthene-9-carboxamide (13), is slightly more selective for HDAC7 with an IC(50) of 0.05muM. Substitution of 6 allows for the modulation of selectivity and potency amongst the class IIa HDAC isotypes.

Condensed bridgehead nitrogen heterocyclic system: synthesis and pharmacological activities of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives of ibuprofen and biphenyl-4-yloxy acetic acid.

Several 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles were prepared by condensation of 4-amino-5-substituted-3-mercapto-(4H)-1,2,4-triazoles (3a,b) with various substituted aromatic acids and aryl/alkyl isothiocyanates through a one-pot reaction. These compounds were investigated for their anti-inflammatory, analgesic, ulcerogenic, lipid peroxidation, antibacterial and antifungal activities. Some of the synthesized compounds showed potent anti-inflammatory activity along with minimal ulcerogenic effect and lipid peroxidation, compared to those of ibuprofen and flurbiprofen. Some of the tested compounds also showed moderate antimicrobial activity against tested bacterial and fungal strains.

Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.

UNLABELLED: We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of the Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was evaluated measuring changes in intracellular pH induced by acute Cl- removal/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microscopy, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was not detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 microM) showed no effect on the basal activity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus secretin, ACh significantly (P < 0.03) increased the maximal rate of alkalinization after Cl- removal and the maximal rate of recovery after Cl- readmission compared with secretin alone (50 nM), indicating that ACh potentiates the stimulatory effect of secretin on the Cl-/HCO3- exchanger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), by the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)- ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (50 microM), but not by the protein kinase C antagonist, staurosporine (0.1 microM). Intracellular cAMP levels, in isolated rat cholangiocytes, were unaffected by ACh alone, but were markedly higher after exposure to secretin plus ACh compared with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- exchanger activity was individually abolished by two calcineurin inhibitors, FK-506 and cyclosporin A (100 nM). CONCLUSIONS: M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh did not influence the basal activity of the Cl-/HCO3- exchanger, but enhanced the stimulation by secretin of this anion exchanger by a Ca2+-dependent, protein kinase C-insensitive pathway that potentiates the secretin stimulation of adenylyl cyclase. Calcineurin most likely mediates the cross-talk between the calcium and adenylyl cyclase pathways. Since secretin targets cholangiocytes during parasympathetic predominance, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) and ACh (Ca2+) could play a major role in the regulation of ductal bicarbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.

Transcranial magnetic stimulation with acepromazine or dexmedetomidine in combination with levomethadone/fenpipramide in healthy Beagle dogs.

The aim of this study was to evaluate the influence of two sedation protocols on transcranial magnetic motor evoked potentials (TMMEPs) after transcranial magnetic stimulation in medium sized dogs. Onset latencies and peak-to-peak amplitudes, elicited in the extensor carpi radialis and cranial tibial muscles, were analysed in 10 healthy Beagles that received either acepromazine or dexmedetomidine in combination with levomethadone/fenpipramide, in a crossover design. Similar TMMEP recordings could be made using both sedation protocols at 80-90% stimulation intensity; however, there were significantly shorter onset latencies with the acepromazine-levomethadone/fenpipramide protocol at 100% stimulation intensity. Reference values were established and it was concluded that both drug combinations are feasible for measuring TMMEPs in medium sized dogs.

Influence of SKF-525A congeners, strophanthidin and tissue-culture media on desensitization in frog skeletal muscle.

1 Microelectrodes have been used to follow changes in membrane potential at end-plate regions of frog skeletal muscle fibres exposed to carbachol; the depolarizing drug was applied to narrow strips of muscle in a rapidly flowing solution containing relatively impermeant anions rather than chloride.2 During prolonged applications of carbachol (10 to 20 muM), the depolarization caused by the drug showed a gradual decline which was attributed to desensitization.3 Desensitization was little if at all affected by supplementing the external solution with factors present in tissue-culture media, or by treating the muscle with strophanthidin (25 muM).4 The rate of repolarization in the presence of carbachol (10 to 20 muM) was greatly increased by the SKF-525A congeners pipenzolate bromide (10 muM) and adiphenine hydrochloride (1 muM). The desensitization-enhancing action of these compounds is discussed.

Functional role of muscarinic M(2) receptors in alpha,beta-methylene ATP induced, neurogenic contractions in guinea-pig ileum.

1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.

Selective blockade of M2 and M3 muscarinic receptors by hexahydrobenzyl-fourdapine and a comparison with zamifenacin.

1. 4-Diphenylacetoxy-N-cyclohexylmethyl-piperidine HCl (hexahydro-benz-4DAP) is more active as an antagonist of carbachol at receptors in guinea-pig isolated ileum, log K (pA2) = 6.64 +/- 0.14 (s.e. 7 results), than at receptors in guinea-pig isolated atria, log K = 5.43 +/- 0.14 (7). In the presence of neostigmine bromide (0.2 microM) the value for atria was 5.62 +/- 0.19 (4), so the lower activity on atria cannot be attributed to hydrolysis of the compound by cholinesterases present in this tissue. 2. The limit of solubility of the free base in Krebs solution (pH 7.6) is about 50 microM for both hexahydrobenz-4DAP and benzyl-fourdapine (benz-4DAP). 3. In experiments on guinea-pig isolated ileum with hexahydro-benz-4DAP given together with 4-DAMP methobromide, the combined dose-ratio was consistent with competition: similar results were obtained with benz-4DAP. 4. In rats anaesthetized with pentobarbitone, hexahydro-benz-4DAP antagonized the effects of bethanechol on blood-pressure in doses that had little effect on heart rate or airflow. There was a limit to the effect which could be obtained, however, and the slopes of the Schild plots were less than one. The effects on rat blood-pressure had a half-life of at least 30 min. 5. In similar experiments with zamifenacin the slopes of the Schild plots were close to 1 and the compound was 10 to 20 times as active on blood-pressure at it was on heart-rate. 6. The limited solubility of the base probably accounts for the flat Schild plots obtained with hexahydro-benz-4DAP, which had about 10 fold selectivity for effects on blood-pressure and was more active than expected from tests on isolated ileum.

Functional and direct binding studies using subtype selective muscarinic receptor antagonists.

1. Muscarinic receptor antagonists were examined in direct binding studies on guinea-pig cardiac and cortical muscarinic receptors. Pirenzepine, dicyclomine and hexahydroadiphenine were shown to be selective ligands for the putative M1-muscarinic receptor. 2. Functional affinity estimates of the muscarinic ligands studied was determined from their ability to inhibit carbachol-stimulated inositol phosphate (IP) accumulation in guinea-pig cortical slices. 3. The affinity estimates for the inhibition of muscarinic agonist-stimulated IP accumulation were better correlated with affinity estimates obtained from binding studies on the M1 than the M2 muscarinic receptor. 4. These data provide additional evidence, both from direct binding and functional studies, for the presence of M1 and M2 muscarinic receptor subtypes.

Actions of some esters of 3,3-dimethylbutan-1-ol (the carbon analogue of choline) on the guinea-pig ileum.

1 Estimates were made of the affinity constants for postganglionic acetylcholine receptors of the guinea-pig ileum of the esters of 3,3-dimethylbutan-1-ol with benzilic, (+/-)-cyclohexylphenylglycollic, (+/-)-mandelic, and diphenylacetic acids.2 Attempts were made to check the competitive nature of the antagonism by using as wide a range of concentrations of antagonist as possible, consistent with their limited solubility, and by testing some of the compunds in the presence of a known competitive antagonist.3 By comparing the affinities with those of the corresponding quaternary nitrogen compounds, the contribution made by the positive charge in the onium group to the binding by receptors may be assessed and has been found to be variable. The carbon analogue of benziloylcholine has about one-tenth of its affinity, that of (+/-)-cyclohexylphenylglycolloylcholine has only about one-sixtieth of its affinity, but that of (+/-)-mandelylcholine has slightly higher affinity than that of (+/-)-mandelylcholine itself.4 3,3-Dimethylbutylacetate appeared to be a partial agonist with an affinity constant of about 2.6 x 10(3). The contribution made by the positive charge to the binding of acetylcholine at these receptors therefore seems likely to lie within the range observed with antagonists and there is no reason to believe that there is necessarily greater intimacy of association by agonists than by antagonists.5 Although the C-C and /#/N-C bonds in -CMe(3) and -/#/NMe(3) are similar in length, the groups do not occupy the same volume in solution in water.

Selective inactivation of muscarinic M2 and M3 receptors in guinea-pig ileum and atria in vitro.

1. The role of muscarinic M2 and M3 receptors in ileal smooth muscle has been evaluated by use of selective receptor alkylation. The alkylating agents, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP mustard) was studied for effects against (+)-cis-dioxolane, at muscarinic M2 and M3 receptors in guinea-pig atria or ileum, respectively. 4-DAMP mustard (10 nM, 40 min exposure) did not discriminate between these muscarinic receptors. In ileum, 4-DAMP mustard, at 100 nM, resulted in a large dextral shift (197 fold) and depression in maxima. In atria there was a smaller dextral shift (14 fold) but no depression in maxima. 2. The muscarinic antagonists, atropine (non-selective), methoctramine (M2-selective) and para-fluorohexahydro-siladiphenidol (pFHHSiD; M3 selective) were studied in protection studies against alkylation by phenoxybenzamine. Washout studies following equilibration of the tissues with atropine (30 nM), methoctramine (0.3 microM) or pFHHSiD (3 microM), showed the compounds to be reversible. No temporal changes in sensitivity to (+)-cis-dioxolane were observed. 3. Exposure, for 20 min, of atria and ileum to phenoxybenzamine (3 and 10 microM respectively) caused dextral shifts and depressions in the maxima of the concentration-response curve to (+)-cis-dioxolane. These effects were inhibited by prior equilibration with atropine (30 nM) and methoctramine (0.1 microM) in atria or atropine (30 nM) and pFHHSiD (3 microM) in ileum. Similar results in ileum were obtained when pilocarpine was used as the agonist. 4. These data were consistent with muscarinic M2 receptors mediating responses in atria and M3 receptors mediating responses in ileum. No evidence was provided for a direct role of muscarinic M2 receptors in ileal contraction.5. It is concluded that receptor protection by reversible antagonists for muscarinic M2 or M3 receptors provides a means to isolate pharmacologically a single subtype in a tissue possessing heterogeneous populations. This technique may prove useful in defining the role of the respective subtypes in smooth muscle contraction.

Inactivation of brain cortex muscarinic receptors by 4-diphenylacetoxy-1-(2-chloroethyl) piperidine mustard.

We demonstrated in this study that 4-DAMP [4-diphenylacetoxy-1-(2- chloroethyl) piperidine] mustard, which cyclizes to the aziridinium ion, behaved as a non-selective, non-competitive inhibitor of muscarinic receptors in rat brain cortex. It inactivated to the same extent the M1, M2 and M4 muscarinic receptors present in this tissue, as well as receptors accessible or not accessible to quaternary antimuscarinic drugs. Under mild incubation conditions, the muscarinic receptors in a state with super high affinity for agonists (SH receptors) were less affected by preactivated 4-DAMP mustard than the receptors in the states with lower affinity for agonists (H and L receptors).

Muscarinic M2 receptor-mediated contraction in the guinea pig Taenia caeci: possible involvement of protein kinase C.

Contraction of the guinea pig taenia caeci is mediated by muscarinic M3 receptors; however, they comprise only 30% of the muscarinic receptors present. This study investigated the role of the predominant M2 receptor population in contractions and possible second messengers involved after M3 receptors were selectively alkylated by 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (60 nM) in the presence of otenzepad (AF-DX 116; 1 microM). Concentration-response curves to oxotremorine-M (oxo-M) in the presence of histamine and isoprenaline were performed in the presence of otenzepad (1 and 3 microM), resulting in a mean apparent pK(B) of 6.49, indicative of an M2 response. As the taenia has intrinsic tone, precontraction with histamine was not necessary and, therefore, in some experiments only isoprenaline was included. In these studies, an M3 response to oxo-M was observed, as the mean apparent pK(B) for otenzepad was 5.89. To investigate protein kinase C (PKC) involvement in the M2 response following M3 inactivation, the inhibitor chelerythrine (1 microM) was included with histamine and isoprenaline in the absence and presence of otenzepad. The oxo-M concentration-response curve was shifted by otenzepad with an apparent pK(B) value of 6.05, a value significantly different from that seen in the absence of chelerythrine (P < 0.05). These results suggest that activation of PKC by a spasmogen such as histamine is necessary to see an M2 response following M3 receptor inactivation.

Comparison of adiphenine and TRH effects on TSH release by rat pituitary in vitro.

The mechanism of action of adiphenine on in vitro rat anterior pituitary TSH release was compared to that of the physiological stimulator TRH. The comparative study showed that adiphenine and TRH were able to increase TSH release in a dose-dependent manner, had similar time courses of action for equipotent stimulating concentrations and produced similar aspects of stimulated TSH cells. However, there were several differences between the effects of adiphenine and TRH. Adiphenine action was inhibited by 20 mM K+; was not calcium dependent; was inhibited by neither thyroid hormones nor somatostatin; was little affected by energy depression. It is concluded that adiphenine probably acts near the ultimate steps of the TSH release pathway and could be a useful pharmacological tool for studying the mechanism of TSH release.

Role of muscarinic M2 and M3 receptors in guinea-pig trachea: effects of receptor alkylation.

Muscarinic M2 receptors account for more than half the muscarinic receptor population in smooth muscles of a number of species and yet it is the smaller M3 receptor population that mediates contraction of many of these tissues. The role of the majority of M2 receptors in the control of smooth muscle tone is unclear. In guinea-pig ileal smooth muscle, an indirect contractile role (re-contraction) for M2 receptors has been demonstrated in tissues subjected to M3 receptor alkylation and stimulation of adenylyl cyclase. The present studies have employed the technique of irreversible receptor alkylation in order to investigate the role of muscarinic M2 and M3 receptors in the control of guinea-pig tracheal smooth muscle tone. Experiments were performed to determine (i) whether an indirect contractile role for M2 receptors can be demonstrated in tracheal smooth muscle as described for ileum, and (ii) whether stimulation of M2 receptors can inhibit isoprenaline-induced relaxations of histamine pre-contracted trachea after selective M3 receptor alkylation. Our results suggest (i) that there is no evidence of M2 receptor-mediated re-contraction of tracheal smooth muscle after M3 receptor alkylation and stimulation of adenylyl cyclase, but (ii) that activation of M2 receptors, after M3 receptor alkylation, has a small inhibitory effect on relaxant responses to isoprenaline in guinea-pig tracheal smooth muscle. Therefore, it appears that the major role of postjunctional muscarinic M2 receptors in guinea-pig trachea remains to be determined.

Rapid evaluation of the efficacy of pharmacologic agents and their analogs in enhancing bladder capacity and reducing the voiding frequency.

We report here the results of a simplified screening method to rapidly compare the pharmacologic action of drugs relevant to the urinary tract. This method avoids the use of anesthesia and of external infusion into the bladder relying on the physiological stimulation of the volume-evoked micturition reflex (VEMR) by diuresis. Mature female rats weighing 240-310 g were placed in restrainer cages that afforded access to food and water but limited movement. Under the rear of the rat, a collecting funnel and weight measuring device was secured. For each VEMR, the weight of the volume voided was recorded on a polygraph which also provided a record of the time of voiding. To evaluate the relative pharmacologic efficacy of the drug under study, 1 mg/kg furosemide along with the drug to be evaluated was diluted in 5 ml of saline and injected subcutaneously. Rats received approximately equimolar concentrations of thiphenamil and 15 other analogs, Ca2+ channel blockers, and K+ channel openers. The furosemide was given to obtain a controlled level of diuresis and avoid the effects of circadian variations in urine flow. Parameters considered were 1) mean volume voided per VEMR, 2) frequency, 3) output, and 4) latency. This model allows the rapid evaluation of drugs designed to increase bladder capacity and decrease the frequency of voiding, and it is particularly useful in evaluating the relative efficacy of drugs that are chemical analogs.

The interaction of 4-DAMP mustard with subtypes of the muscarinic receptor.

The compound 4-DAMP mustard (N-2-chloroethyl-4-piperidinyl diphenylacetate) is a 2-chloroethylamine derivative of the selective muscarinic antagonist 4-DAMP (N,N-dimethyl-4-piperidinyl diphenylacetate). At neutral pH, 4-DAMP mustard cyclizes spontaneously into an oziridinium ion that binds covalently with muscarinic receptors. Analysis of the kinetics of receptor alkylation showed that the interaction of 4-DAMP mustard with M2 and M3 receptors was consistent with a model in which the aziridinium ion rapidly forms a reversible complex with the receptor which converts to a covalent complex at a relatively slower rate. The rate constant (k2) for alkylation of M2 and M3 receptors was approximately the same (k2 = 0.1 min-1); however, the affinity of the aziridinium ion for the M3 receptor (KD = 7.2 nM) was approximately 6.3-fold greater than that for the M2 receptor (KD = 43 nM). The results of competitive binding experiments on Chinese hamster ovary cells transfected with the M1 - M5 subtypes of the muscarinic receptor showed that the affinity of the aziridinium ion for the M1, M3, M4 and M5 subtypes was approximately the same and about 11-fold greater than that for the M2 receptor. 4-DAMP mustard is a useful tool for selectively inactivating all non-M2 muscarinic receptors, particularly when it is used in the presence of a reversible M2 selective antagonist to protect the M2 receptor from alkylation. The results of studies on isolated smooth muscle preparations that have had their M3 receptors alkylated with 4-DAMP mustard are consistent with the postulate that the M2 receptor can elicit contraction by inhibiting the relaxant effect of isoproterenol and forskolin on histamine induced contractions.

The use of irreversible ligands to inactivate receptor subtypes: 4-DAMP mustard and muscarinic receptors in smooth muscle.

Irreversible ligands are useful tools for investigating the function of receptor subtypes in various physiological processes. The mechanism for alkylation involves the formation of a reversible receptor complex followed by a covalent reaction. The extent of receptor alkylation is determined by the dissociation constant of the reversible complex and the rate constant for conversion to the covalent complex. Selectivity can be achieved if the irreversible ligand exhibits a difference in its dissociation constants for receptor subtypes. Selective alkylation can also be achieved using a selective competitive inhibitor to protect the desired receptor subtype. By using the non-M2-selective irreversible antagonist, 4-DAMP mustard, in combination with the competitive M2-selective antagonist, AF-DX 116, it has been possible to achieve a highly selective inactivation of all non-M2 subtypes of the muscarinic receptors in smooth muscle and has enabled the discovery of the functional role of M2 receptors in smooth muscle.