Palauans who chew betel nut: social impact of oral disease.

BACKGROUND: Chewing betel nut is a tradition extending from Southeast Asia to the Pacific. Globally, betel nut is the fourth main psychotropic substance containing a stimulant, arecoline, that has a similar effect to nicotine. In Palau, there is broad acceptance of betel nut chewing. One of the largest immigrant groups in Hawaii is the Palauans. Chewing betel nut has significant social implications that make it difficult for those who engage in this practice to separate potential oral disease from the social importance. However, little is known about the social impact of oral disease from chewing betel nut on Palauans in Hawaii. AIM: The study aimed to describe the perceptions of betel-chewing Palauans in Hawaii regarding betel nut and to determine the social impact of oral disease among these individuals. METHODS: Descriptive study conducted on the island of Oahu, Hawaii with 30 adult Palauans. Data were collected using the Oral Health Impact Profile-14 to measure perceptions of social impact of oral disease on well-being. Demographic and general health information was collected. RESULTS: Participants perceived little negative social impact of oral disease on well-being. DISCUSSION: Families, peers and society exert a strong influence on the decision to chew betel nut, a known carcinogen. Participants in this study showed little concern on the impact of betel nut chewing on their oral health. They continue the habit in spite of the awareness of potential for oral disease. IMPLICATIONS FOR NURSING AND HEALTH POLICY: Nurses face challenges in educating Palauans about the negative aspects of betel nut, particularly those related to oral health especially when they do not perceive problems. Nurses must be involved in the development of health policies to design and implement strategies to promote behavioural change, and to ensure clinical services that are culturally sensitive to betel nut chewers.

Stimulation of cyclic AMP accumulation and phosphoinositide hydrolysis by M3 muscarinic receptors in the rat peripheral lung.

The effects of oxotremorine-M (oxo-M), a muscarinic agonist, on cyclic AMP (cAMP) accumulation in slices of the rat peripheral lung were investigated. Oxo-M stimulated cAMP accumulation in a concentration-dependent manner with an EC50 value of 4.2 microM and a maximal effect of 2.4 +/- 0.39-fold over basal. In the presence of forskolin (25 microM), the maximal effect of oxo-M was increased to 14.1 +/- 4.0-fold over basal. Forskolin alone caused a 5.9 +/- 2.2-fold increase in cAMP relative to basal; therefore, the combination of both drugs was more than additive. The effects of oxo-M on cAMP accumulation were unaffected by tetrodotoxin, indicating that the action of oxo-M was not mediated by neuronal release of neurotransmitters. Oxo-M had a small inhibitory effect on cAMP in a homogenate preparation, indicating that the stimulatory response to oxo-M in slices of the lung is not due to direct stimulation of adenylyl cyclase. Characterization of the oxo-M potentiation of forskolin-stimulated cAMP accumulation using different muscarinic antagonists yielded calculated pKB values that agreed with binding affinities for the M3 subtype. Oxo-M elicited phosphoinositide hydrolysis in the lung, and the nature of the antagonism of this response was also consistent with that expected for an M3-mediated response. cAMP accumulation in the presence of oxo-M (100 microM), forskolin (12 microM), or both drugs combined was inhibited by indomethacin (1 microM). These results demonstrate that the M3 receptor stimulates cAMP accumulation and phosphoinositide hydrolysis in the rat peripheral lung, and the mechanism for cAMP stimulation may involve arachidonic acid metabolites.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a serum-free chemically defined medium: effects on acute and chronic opiate regulation of adenylate cyclase activity.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.

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.

Modulation of adenylate cyclase activity by a cytosolic factor following chronic opiate exposure in neuroblastoma x glioma NG108-15 hybrid cells.

A soluble cytosolic factor from neuroblastoma x glioma NG108-15 hybrid cells stimulates adenylate cyclase activity in isolated membrane preparations. This cytosolic component is heat stable, pronase insensitive, has a molecular weight less than 350 daltons and an absorbance peak at 260 nm. The stimulation is immediate, independent of Ca++ and exhibits a sigmoidal concentration dependency curve. The cytosolic factor stimulated adenylate cyclase activity in etorphine treated cells (100 nM etorphine, 16 hrs) to a greater extent than in control cells. In addition, cytosolic factor derived from etorphine treated cells, as compared to control cells, displayed an increased capacity to stimulate adenylate cyclase. It is suggested that the observed cytosolic factor may be adenosine and that cells chronically treated with an opiate exhibit an increase in both concentration and sensitivity to this agent.

AIDS drugs & the pharmaceutical industry: a need for reform.

The pharmaceutical industry has long enjoyed substantial profits despite increased requirements for drug approval and various attempts to regulate the industry. Drug companies have avoided effective regulation by blaming high prices on the costs of research and development. The search for drugs effective in combatting HIV and AIDS related illnesses has provided a stark background on which to view the actions and justifications of drug companies. Despite increased cooperation between government and the drug industry and expedited approval of several useful drugs, these drugs are still prohibitively expensive. This Article explores the history and economics of the drug industry and proposes a system of national price regulation for all drugs.

Attitudes toward genetic research review: results from a survey of human genetics researchers.

BACKGROUND: Researchers often relate personal experiences of difficulties and challenges with Institutional Review Board (IRB) review of their human genetic research protocols. However, there have been no studies that document the range and frequency of these concerns among researchers conducting human genetic/genomic studies. METHODS: An online anonymous survey was used to collect information from human genetic researchers regarding views about IRB review of genetic protocols. Logistic regression was used to test specific hypotheses. Results from the national online survey of 351 human genomic researchers are summarized in this report. RESULTS: Issues involving considerable discussion with IRBs included reconsent of subjects (51%), protection of participants' personal information (39%) and return of results to participants (34%). Over half of the participants had experienced one or more negative consequences of the IRB review process and approximately 25% had experienced one or more positive consequences. Respondents who had served on an IRB were about 80% more likely to report positive consequences of IRB review than their colleagues who had never served on an IRB (p = 0.03). Survey responses were mixed on the need for reconsent before data sharing and risks related to participant reidentification from genomic data. CONCLUSION: The results from this study provide important perspectives of researchers regarding genetic research review and show lack of consensus on key research ethics issues in genomic research.

Specific inhibition of isoproterenol-stimulated cyclic AMP accumulation by M2 muscarinic receptors in rat intestinal smooth muscle.

The ability of oxotremorine-M to inhibit cyclic AMP accumulation in the presence of a variety of adenylate cyclase activators was studied in slices from the longitudinal muscle of the rat ileum. Oxotremorine-M was found to inhibit forskolin- and isoproterenol-stimulated cyclic AMP accumulation maximally by 17 and 32%, respectively, but not the stimulation due to other activators of adenylate cyclase. Inhibition of cyclic AMP accumulation by oxotremorine-M was unaffected by tetrodotoxin and was completely reversed by atropine. AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidynyl]acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one) an M2-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant (KB) of 0.20 microM, consistent with the dissociation constants for binding at the M2 muscarinic receptor site (KD = 0.092 microM) and inhibition of adenylate cyclase activity (KB = 0.13 microM). Hexahydrosiladifenidol, an M3-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant of 0.67 microM, again consistent with the dissociation constant for binding at the M2 site (KD = 0.83 microM). The agreement between the estimates of the dissociation constants of muscarinic antagonists for binding and for inhibition of cyclic AMP accumulation suggest that oxotremorine-M inhibition of isoproterenol-stimulated cyclic AMP accumulation in slices of rat intestinal smooth muscle is mediated by the M2 receptor.

Involvement of both inhibitory and stimulatory guanine nucleotide binding proteins in the expression of chronic opiate regulation of adenylate cyclase activity in NG108-15 cells.

Chronic etorphine treatment of neuroblastoma X glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (Ni) and stimulatory (NS) components. Inactivation of Ni by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of NS, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E1, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through Ni resulting in the unimpeded expression of NS activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.

Effect of phospholipases on chronic opiate action in neuroblastoma X glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.

Kinetics of activation and in vivo muscarinic receptor binding of N-(2-bromoethyl)-4-piperidinyl diphenylacetate: an analog of 4-DAMP mustard.

4-DAMP mustard (N-2-chloroethyl)-4-piperidinyl diphenylacetate) has been shown to selectively and irreversibly inhibit muscarinic receptors. In an attempt to increase the rate of formation and peak concentration of the reactive intermediate, an analog [N-(2-bromoethyl)-4-piperidinyl diphenylacetate (4-DAMP bromo mustard)] was synthesized and the molecular formula confirmed by mass analysis. The 4-DAMP bromo mustard was shown to cyclize in phosphate buffer (pH 7.4) to the corresponding aziridinium ion with a first-order rate constant (k1) of 0.071 min-1 at 0 degrees C. At 25 degrees C and 37 degrees C, the formation of the aziridinium ion was nearly instantaneous (100% cyclized within 15 sec) at neutral pH. The rate constants (k2) for the hydrolysis of the aziridinium ion at 25 degrees C and 37 degrees C (pH 7.4) were 0.0027 and 0.010 min-1, respectively, in excellent agreement with the published rate constants for the hydrolysis of the aziridinium ion formed from 4-DAMP mustard. In vivo treatment with 4-DAMP bromo mustard in rats resulted in irreversible inhibition of muscarinic receptor binding in peripheral, but not central nervous system, tissues, suggesting that the quickly formed aziridinium ion does not penetrate the blood-brain barrier.

Diffuse alveolar haemorrhage associated with progressive systemic sclerosis.

A 41 year old man with an eight year history of progressive systemic sclerosis developed severe diffuse alveolar haemorrhage and died. The importance of diffuse alveolar haemorrhage as a rare but potentially serious complication of connective tissue disease should not be overlooked.

The interaction of the enantiomers of aceclidine with subtypes of the muscarinic receptor.

The pharmacological activity of the enantiomers of aceclidine was investigated in Chinese hamster ovary cells transfected with the M1 through M5 subtypes of the muscarinic receptor and also in the rat heart and parotid gland that express primarily M2 and M3 receptors, respectively. When measured by stimulation of phosphoinositide hydrolysis in Chinese hamster ovary cells transfected with the M1, M3 and M5 muscarinic subtypes, the potency of S-(+)-aceclidine was approximately 2- to 4-fold greater than that of R-(-)-aceclidine, whereas the maximal response of the R-(-)-isomer was only 44 to 64% that of the S-(+)-isomer. When measured by inhibition of forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the M2 and M4 muscarinic subtypes, the potency of S-(+)-aceclidine was approximately 3.5-fold greater than that of R-(-)-aceclidine. In cells transfected with the M2 muscarinic receptor, the maximal responses of the enantiomers were the same, whereas the maximal response of R-(-)-aceclidine was 86% that of S-(+)-aceclidine in cells transfected with the M4 muscarinic subtype. The activities of the enantiomers of aceclidine at native M2 and M3 muscarinic receptors coupled to inhibition of adenylyl cyclase activity in the heart and stimulation of phosphoinositide hydrolysis in the parotid gland, respectively, were similar to those observed in Chinese hamster ovary cells transfected with the corresponding receptor subtypes. We devised a simple quantitative method for using our data in Chinese hamster ovary cells to predict the relative potencies of agonists in a more sensitive assay in which the agonists produce a full maximum response. By using this method, we were able to predict the relative potencies of the enantiomers for eliciting contractions in the guinea pig ileum, an M3 muscarinic response, from their activity in Chinese hamster ovary cells transfected with the M3 muscarinic subtype. Our method of analysis should have application in a variety of studies in which transfected cells are used to determine the pharmacological activity of agonists.

The quaternary transformation products of N-(3-chloropropyl)-4-piperidinyl diphenylacetate and N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) have differential affinity for subtypes of the muscarinic receptor.

A 3-chloropropylamine derivative (N-(3-chloropropyl)-4-piperidinyl diphenylactate) of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate was synthesized and its conversion to a stable azetidinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4, N-(3-chloropropyl)-4-piperidinyl diphenylactate formed a stable azetidinium ion with a half-time of approximately 3.6 hr. The selectivity of the azetidinium ion for native M1, M2 and M3 subtypes of the muscarinic receptors was investigated in competitive binding experiments on the hippocampus, heart and submaxillary gland of rats, respectively, using N-[3H]methylscopolamine as the radioligand. The azetidinium ion exhibited equivalent high affinities for the M1 and M3 mucarinic receptor subtypes (KD = approximately 5 nM), but 10-fold lower affinity for the M2 muscarinic receptor subtype (KD = 44 nM). Similar competitive binding experiments were carried out on Chinese hamster ovary cells transfected with the M1 through M5 subtypes of the muscarinic receptor. In these experiments, the azetidinium ion exhibited similar high affinities for the M1, M3, M4 and M5 muscarinic receptor subtypes (KD = approximately 2.4 nM), but approximately 14-fold lower affinity for the M2 muscarinic receptor subtype (KD = 34 nM). In contrast to the azetidinium ion, the parent N-(3-chloropropyl)-4-piperidinyl diphenylactate compound was 130-fold less potent. An analogous series of experiments were carried out with the aziridinium ion derived from the muscarinic receptor alkylating agent, N-(2-chloroethyl)-4-piperidinyl diphenylactate. For these binding experiments, the incubations were carried out at 0 degrees C to prevent the aziridinium ion from alkylating muscarinic receptors. The aziridinium ion was found to have equivalent high affinities for the M1, M3, M4 and M5 subtypes of the muscarinic receptor (KD = approximately 6.6 nM), but about 11-fold lower affinity for the M2 muscarinic receptor subtype (KD = 72 nM). Our results suggest that 3-haloalkylamine derivatives of 4-piperidinyl diphenylactate may be candidate prodrugs that may penetrate into brain and form azetidinium ions that have a long-lasting central anticholinergic effect.

Acetylcholine-induced desensitization of muscarinic contractile response in Guinea pig ileum is inhibited by pertussis toxin treatment.

We investigated the effects of pertussis toxin treatment on acetylcholine-induced desensitization of the muscarinic contractile response in guinea pig ileum. Incubation of the isolated ileum with acetylcholine (30 microM) for 20 min caused a decrease in the sensitivity of the ileum to the contractile action of the muscarinic agonist oxotremorine-M. This desensitization was characterized by an increase in the EC(50) value of oxotremorine-M without a change in its maximal effect. A maximal 4- to 5-fold increase in the EC(50) value of oxotremorine-M was measured at the earliest time investigated after acetylcholine treatment (5 min), and normal sensitivity recovered within approximately 20 min after washout of acetylcholine. Treatment of the ileum with pertussis toxin caused a small increase in the contractile response to oxotremorine-M when measured without prior exposure to acetylcholine. After exposure to acetylcholine, little desensitization was observed in ilea that had been treated with pertussis toxin. Pertussis toxin-treatment caused a small increase in oxotremorine-M-mediated phosphoinositide hydrolysis and a large decrease in oxotremorine-M-mediated inhibition of forskolin-stimulated cAMP accumulation in slices of the longitudinal muscle of the ileum. Exposure of the ileum to acetylcholine had no desensitizing effect on the ability of oxotremorine-M to elicit phosphoinositide hydrolysis, indicating that the mechanism for desensitization of the contractile response occurs at a level downstream from the receptor and phosphoinositide hydrolysis. Our results suggest that activation of muscarinic receptors coupled to pertussis toxin-sensitive G(i) and G(o) is required for most of the desensitization observed in this study.

A simple method for estimation of agonist activity at receptor subtypes: comparison of native and cloned M3 muscarinic receptors in guinea pig ileum and transfected cells.

We describe a simple method for calculating the pharmacological activity of an agonist (A) relative to a standard agonist (S) using only the concentration-response curves of the two agonists. In most situations, we show that the product of the ratios of maximal responses (Emax - A/Emax - S) and potencies (EC50 - S/EC50 - A) is equivalent to the product of the affinity and intrinsic efficacy of A expressed relative to that of S. We refer to this term as the IRA value of A. In a cooperative system where the concentration-response curve of the standard agonist is steep and that of the test agonist is flatter with a lower maximal response, the simple calculation of IRA described above underestimates agonist activity; however, we also describe a means of correcting the IRA in this situation. We have validated our analysis with modeling techniques and have shown experimentally that the IRA values of muscarinic agonists for stimulating contractions in the guinea pig ileum (M3 response) are in excellent agreement with those measured in the phosphoinositide assay on Chinese hamster ovary cells expressing the M3 muscarinic receptor.

Attenuation of enkephalin activity in neuroblastoma X glioma NG108-15 hybrid cells by phospholipases.

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.

Biodistribution of a novel antiestrogen (Analog II) in the mouse and rat.

The biodistribution of a novel antiestrogen Analog II was determined in the mouse and rat. The tritiated product, [3H]-Analog II was prepared by New England Nuclear and was purified by preparative chromatography using silica gel and petroleum ether/methylene chloride (80:20). The fat tissue had the highest uptake due to the hydrophobic nature of Analog II. The second highest uptake was in the mouse uterine tissue which was greater than that observed in the rat. The differences in biodistribution between the mouse and rat may partially explain the differences in biological activity of Analog II previously observed in these two animal species.

Synthesis and biological evaluation of a series of gem-dichlorocyclopropanes as antitumor agents.

As part of our continuous effort to produce non-steroidal antiestrogens demonstrating less intrinsic estrogenicity and greater antagonism than those in use, a series of Analog II (1,1-dichloro-2,3-diphenylcyclopropanes) derivatives was synthesized. The compounds were tested for their ability to inhibit the growth-stimulating action of estradiol in the immature mouse uterus and estrogen receptor (ER) (+) MCF-7 human breast cancer cells in vitro. Like Analog II, the derivatives were found to have no intrinsic estrogenicity (except 30) and they antagonized estradiol action less completely than the lead compound. Polarity improved the ER binding affinity of Analog II, but was quite small for all compounds, except 30, for which it was comparable to tamoxifen. Six compounds (8, 10, 14, 23, 29 and 30) demonstrated antiproliferative activity toward MCF-7 cells, in vitro, and the mean inhibition period over 6 days ranged from 20 to 37%. Only compound 30 was reversed by estradiol.

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.