Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer.

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are "gold standard" because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the "mix & measure" assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca+2 measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compared the IC50 values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca+2 mobilization assays can be an alternate to radioligand binding assays.

Learning deficits induced by 4 belladonna alkaloids are preferentially attenuated by tacrine.

AIM: To examine the antagonism of tacrine on the amnesic effects of scopolamine (Sco), anisodine (AT3), atropine (Atr), and anisodamine (Ani). METHODS: Cognitive functions and locomotor activities were determined using two sessions of step-through and open-field tests, respectively. Mice were injected with one of the belladonna alkaloids (0.05-50 mumol.kg-1, i.p.) and tacrine (50 mumol.kg-1, s.c.) 30 min before the first session. RESULTS: Tacrine completely blocked the avoidance-learning deficit caused by Sco 0.5 mumol.kg-1, AT3 and Atr 5 mumol.kg-1, or Ani 50 mumol.kg-1. But tacrine partly antagonized the learning deficit induced by Sco 5-50 mumol.kg-1 or Atr and AT3 50 mumol.kg-1. The avoidance-memory deficit caused by Sco 0.05-5 mumol.kg-1 or Atr 5 mumol.kg-1 was completely or partly attenuated by tacrine, which did not antagonize the memory deficit elicited by Sco and Atr 50 mumol.kg-1, AT3 5 and 50 mumol.kg-1, and Ani 50 mumol.kg-1. During the acquisition, the locomotor activity of the mice was inhibited by tacrine. This reduction was completely antagonized by Sco 0.5-50 mumol.kg-1, AT3 5-50 mumol.kg-1, Atr 5-50 mumol.kg-1, and only partly antagonized by AT3 and Atr 0.5 mumol.kg-1 or Ani 50 mumol.kg-1. CONCLUSION: Compared with the avoidance-memory deficit, the avoidance-learning deficit caused by belladonna alkaloids is more preferentially attenuated by tacrine.

Identification of drugs competing with d-tubocurarine for an allosteric site on cardiac muscarinic receptors.

d-Tubocurarine behaved as a weak allosteric inhibitor of N-[3H] methylscopolamine binding to cardiac M2 muscarinic receptors. In a low ionic strength buffer devoid of bivalent ions, d-tubocurarine recognized cardiac M2 receptors in the micromolar concentration range and decreased their affinity for N-[3H]methylscopolamine by at most 4-fold. To identify the compounds that preferentially recognize this accessory site (as opposed to the classical muscarinic binding site), we measured the inhibition by different drugs of N-[3H]methylscopolamine binding, in the absence or presence of d-tubocurarine. The effect of gallamine was competitively inhibited by d-tubocurarine; both drugs compete for the same accessory site on muscarinic receptors. The effects of dexetimide, levetimide, 4-diphenylacetoxy-N-ethylpiperidine ethobromide, AF-DX 116, and telenzepine on N-[3H]methylscopolamine binding were not affected or were barely affected by d-tubocurarine; these compounds preferentially recognize another binding site (probably the muscarinic binding site). The dose-effect curves for pentamethylene-bis(4-diphenylacetoxymethylpiperidine) bromide and methoctramine were shifted, but at most 10-fold, by d-tubocurarine. It is likely that (in this low ionic strength incubation buffer) methoctramine and pentamethylene-bis(4-diphenylacetoxymethylpiperidine)bromide had comparable affinities for the muscarinic site and the accessory site. d-Tubocurarine competitively inhibited their binding to the accessory site and allosterically inhibited their binding to the muscarinic site. This resulted in a large decrease (40-60-fold) of their overall affinity for muscarinic receptors.

M3 muscarinic acetylcholine receptor coupling to PLC in rat exorbital lacrimal acinar cells.

This study was designed to characterize the muscarinic acetylcholine receptor (mAChR) subtype present in rat exorbital lacrimal gland as well as its biochemical coupling. The nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) binds with high affinity to a homogeneous population of binding sites in both membranes [dissociation constant (Kd) = 82.3 +/- 3.2 pM] and acinar cell (Kd = 170.3 +/- 20 pM) preparations. Muscarinic antagonist inhibition of [3H]NMS binding is homogeneous with the following order of potency: atropine > or = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > pirenzepine > 11-([2-(diethylamino)-ethyl]-1-piperidinyl)-acetyl- 5,11-dihydro-6H-pirido[2,3-b]1,4,benzo diazepine-6-one (AFDX 116). Both the affinity of the selective antagonists 4-DAMP, pirenzepine, and AFDX 116 and Northern blot analysis of lacrimal gland mRNAs show a single mAChR population of the M3 subtype. Muscarinic agonist inhibition of [3H]NMS binding displays both high (approximately 20%)- and low-affinity sites (approximately 80%). Both the receptor occupancy and the stimulation by agonists or the inhibition by antagonists of the accumulation of [3H]inositol phosphate were examined under identical conditions with respect to tissue preparations (acinar cells) and buffer (Krebs-Ringer). Results demonstrate 1) the efficient coupling of the M3 mAChR subtype with the phosphatidylinositol (4,5))bisphosphate-specific phospholipase C activity and 2) that the efficacy of a muscarinic agonist is dependent on its structure. Lastly, comparison of the agonists affinity and potency to trigger the [3H]inositol phosphate accumulation suggests that the occupation of the high-affinity agonist binding state of the M3 mAChR was involved in the cellular response.

Characterization of muscarinic cholinergic receptors in intact myocardial cells in vitro.

Muscarinic acetylcholine receptors (mAChR) were studied on heart cells grown in culture by the radioligand binding technique. We used [3H]n-methyl-scopolamine to monitor the level of receptors on intact cardiocytes. The number of mAChR was very low during the first days in culture (23 fmol/dish). It increased gradually until it reached a plateau on the 4th day (180 fmol/dish), where it remained for 1-2 weeks. To determine whether contractile activity affected the level or affinity of mAChR, the cardiocytes were exposed to agents that stimulate or arrest the heart beat. Treatment with triiodothyronine (T3, 10-90 nM) for 48 hr caused a reduction in the level of the receptors by 20-30% without changing significantly the affinity of the receptors. Similarly, electrical stimulation caused a reduction in the level of the receptors by 30-40%, without a significant influence on creatine kinase activity. When the myocardial cells were treated with Ca-channel blocker such as metoxyverapamil (D600) (10-30 micrograms/mL) or diltiazem (10-25 micrograms/mL) the level of the receptors was also reduced by 30-40%. The reduction in the receptor binding sites was accompanied by an increase in Kd from 0.8 to 3.2 nM in D600-treated cells, whereas there was no significant change in the radioligand affinity after application of diltiazem. Treatment with D600 or T3 together with cycloheximide showed that under these experimental conditions the rate of receptor degradation was accelerated. The half-life of the receptors in the control was 27 hr, whereas the half-lives of T3 and D600 were 15 and 18 hr, respectively. It is concluded that regulation of the amount of cholinergic receptors occurs at the level of receptor breakdown, and simple linkage does not exist between the rate of cardiac contractions and the number of mAChR.

Muscarinic receptor subtypes in human and rat colon smooth muscle.

Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8.01 +/- 1.0 microM for human samples and 6.07 +/- 1.1 microM for rat samples. These values are close enough to suggest that the first subtype of muscarinic receptor may be considered cardiac (M2) and the second subtype glandular (M3). The relative densities of the receptor subtypes are significantly different for both species. Human colon samples show the major densities of subtype M2, 22.62 +/- 1.11 fmol/mg protein, this represents 75.66 +/- 3.73% of the total receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues.

A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a significant change in the dissociation constants. The affinity of 4-DAMP mustard and its transformation products for muscarinic receptors was determined in competitive binding experiments with N-[3H] methylscopolamine, and the results show that the aziridinium ion of 4-DAMP mustard was the most potent form, compared with the parent 2-chloroethylamine (4-DAMP mustard) and the alcoholic hydrolysis product. The rates of receptor alkylation by 4-DAMP mustard were measured in the rat heart and gland. Virtually no alkylation (less than 1%) occurred in the heart at a 4-DAMP mustard concentration of 1.6 nM, after 30 min, whereas almost 50% alkylation was observed in the gland under the same conditions. Almost complete alkylation of receptors in the gland could be achieved at a 4-DAMP mustard concentration of 200 nM, after 1 hr. Treatment of the isolated rat ileum with 4-DAMP mustard caused an irreversible blockade of contractions elicited by the muscarinic agonist oxotremorine-M, and this blockade persisted after extensive washing. The results presented here show that 4-DAMP mustard forms an aziridinium ion that binds irreversibly to muscarinic receptors and exhibits selectivity for M3, compared with M2 muscarinic receptors.

Muscarinic receptor subtypes in guinea pig airways.

The muscarinic receptors present in the guinea pig tracheobronchial tree were characterized using ligand-binding studies and functional approaches. The binding constants of four selective antagonists, pirenzepine,[11-([2-[(diethylamino) methyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-pyrido(2,3) (1,4) benzodiazepine-6-on] (AF-DX 116), methoctramine, and 4-diphenylacetoxy-Nmethylpiperidine methobromide(4-DAMP) were examined. Heterogeneity in the airway muscarinic receptors population was revealed by competitive binding experiments against [N-methyl3H]scopolamine with the M2 muscarinic antagonists AF-DX 116 and methoctramine. In guinea pig lung and trachea, AF-DX 116 and methoctramine recognized 86-88% and 50-60% of total receptors with high affinity, respectively. These receptors exhibit binding constants for these two compounds similar to those of the M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors. In lung and trachea, we found no evidence for a high-affinity [N-methyl-3H]pirenzepine binding sites. In functional studies, pirenzepine, methoctramine, and 4-DAMP inhibited the methacholine-induced contraction of lung parenchymal, main bronchial, and tracheal strips with affinities characteristic of smooth muscle M3 receptors. These results are consistent with the presence of M2 and M3 receptors in guinea pig airways. Throughout the airways, the muscarinic receptors mediating smooth muscle contraction are of the M3 subtype.

Functionalized congener approach to muscarinic antagonists: analogues of pirenzepine.

The M1-selective muscarinic receptor antagonist pirenzepine 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) was derivatized to explore points of attachment of functionalized side chains for the synthesis of receptor probes and ligands for affinity chromatography. The analogues prepared were evaluated in competitive binding assays versus [3H]-N-methylscopolamine at four muscarinic receptor subtypes (m1AChR-m4AChR) in membranes from rat heart tissue and transfected A9L cells. 9-(Hydroxymethyl)pirenzepine, 8-(methylthio)pirenzepine, and a series of 8-aminosulfonyl derivatives were synthesized. Several 5-substituted analogues of pirenzepine also were prepared. An alternate series of analogues substituted on the 4-position of the piperazine ring was prepared by reaction of 4-desmethylpirenzepine with various electrophiles. An N-chloroethyl analogue of pirenzepine was shown to form a reactive aziridine species in aqueous buffer yet failed to affinity label muscarinic receptors. Within a series of aminoalkyl analogues, the affinity increased as the length of the alkyl chain increased. Shorter chain analogues were generally much less potent than pirenzepine, and longer analogues (7-10 carbons) were roughly as potent as pirenzepine at m1 receptors, but were nonselective. Depending on the methylene chain length, acylation or alkyl substitution of the terminal amine also influenced the affinity at muscarinic receptors.

Muscarinic receptor stimulation increases inositol-phospholipid metabolism and inhibits cyclic AMP accumulation in PC12 cells.

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)