Rachel Rubinstein

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN‐A‐343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope‐labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN‐A‐343 enhanced the release of isotope‐labeled ACh, whereas oxotremorine inhibited the K+‐evoked release but had no effect on the basal release. The enhancing effect of McN‐A‐343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN‐A‐343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN‐A‐343 was 3.6‐fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6‐fold more potent in the cerebellum. The displacements of radio‐labeled pirenzepine and m‐dioxolane confirmed the M1 subtype preference of McN‐A‐343 and the M2 subtype preference of oxotremorine. These observations suggest that (a) activation of one subpopulation of muscarinic receptors by McN‐A‐343 enhances ACh release, whereas activation of another subpopulation by oxotremorine decreases this process (these subpopulations may correspond to the M1 and M2 subtypes, respectively), (b) the effect of the stimulatory receptors is pronounced at the resting state and that of the inhibitory receptors during depolarization, and (c) activation of the stimulatory receptors may be inversely related to the activity of the Ch transporter.

Histamine-mediated acetylcholine release in the guinea-pig ileum.

The isometric contraction induced by histamine in guinea-pig ileum longitudinal muscle was biphasic in Krebs ([Ca]: 3.36 mM) but monophasic in Tyrode solution ([Ca]: 1.80 mM). The late phase (histamine: 20-400 nM) was common to the two solutions but the early phase (histamine: 2-20 nM) was observed only in Krebs solution. This early phase could be blocked with atropine (0.01-0.2 microM), morphine (0.1, 1 microM), adenosine (5, 20 microM) and tetrodotoxin (0.3 microM) without affecting the late phase. Washout of morphine or adenosine was fast. Neostigmine (100 nM) greatly potentiated the effect of histamine (4 nM) in the early phase, the muscle undergoing almost maximum contraction but also reversible desensitization to doses of histamine less than or equal to 20 nM for as long as 40 min after washout. Beyond this concentration, the preparation responded to increasing doses of histamine as observed in the late phase. It is concluded that low concentrations of histamine that have no observable direct effect on muscle contractility release acetylcholine in the presence of [Ca] 3.36 mM, the early phase being entirely due to release of endogenous acetylcholine.

A comparative study of the affinities of some tricyclic antidepressants for the muscarinic cholinergic receptor in human and guinea-pig bladder, ileum and brain in relation to differential drug potency

Following a report that nortriptyline was found useful in the control of enuresis in adults, presumably as an anticholinergic, its likely mechanism of action and apparent bladder specificity have now been investigated in vitro. The ratios of anticholinergic potencies (reciprocal of dissociation contents, Ki) for four different tricyclic antidepressants, derived from competitive binding assays with (-)[3H]QNB in tissue homogenates, in the order (human) detrusor muscle/ileal longitudinal muscle/caudate, are as follows: Nortriptyline, 5/4/7; desipramine, 2/1/5/; clomipramine, 4/3/27; amitriptyline, 25/14/56. The apparent selective effect of nortriptyline on the bladder cannot be ascribed to its higher affinity to bladder receptors. Still, this drug is the least discriminatory of the four. Hence, at a given concentration, it is expected to affect tissue embodying a low density receptor pool sooner than tissue having a large receptor reserve. The ratios of the densities of (-)[3H]QNB binding sites in the order detrusor muscle/ileal muscle/cortex is 1/3/5, supporting the present contention. In the guinea-pig, the ratios of the anticholinergic potency in the order bladder/proximal ileum/distal ileum/cortex are as follows: Nortriptyline, 25/5/6/33; desipramine, 8/2/2/14; amitriptyline, 100/14/20/100; clomipramine, 17/3/5/33. Also, the ratios of the densities of binding sites are 1/6/5/2. Hence, data derived from assays in the guinea-pig are not representative of those derived from human tissue.

Affinity of nortriptyline to muscarinic receptors in the bladder and ileum of man and guinea-pig

The antagonism by nortriptyline of carbachol- or urecholine-induced contractions was studied in strips of ileum and bladder derived from man and guinea-pig. Analyses of the results by the dose ratio method (Schild plots) showed significant differences in the affinities of the relevant muscarinic receptors to the antagonist: The Ki values in microM were as follows: Human ileum, 0.938; human bladder, 0.298; guinea-pig ileum, 0.159; guinea-pig bladder, 0.333 and 0.453. In man, the higher affinity of the drug to the receptors in the bladder than to those in the ileum may be of consequence in its therapeutic application as an antienuretic agent.

A study of the contractile response to acetylcholine in human ileal and detrusor muscle: origin of the low efficacy of acetylcholine

In Krebs solution (3.36 mM Ca2+), the maximal contractile response of human ileal and urinary bladder detrusor muscle to acetylcholine (ACh) was 40-60% that to carbachol (CCh). The maximum response to ACh was reached at a bath concentration of about 1 microM and was maintained throughout a range extending to 100 microM. In the presence of neostigmine (0.1 microM), the maximum response to ACh reached the level of that of CCh. However, bioassay of bath concentrations of ACh at various points of the maximal response in the absence of neostigmine revealed only slight to insignificant diminution of the applied concentration of ACh. Joint application of ACh and CCh generated a dose-response profile consistent with a model of competitive antagonism between a full agonist (CCh) and a partial one (ACh). Also, choline (100 microM) reduced the maximum response to ACh in the presence of neostigmine and that to CCh to 60-80% of control. These observations are consistent with a mechanism whereby intact cholinesterase together with its substrate ACh and possibly a breakdown product of ACh constitute a filter or diffusional barrier regulating the flow of agonist from the enzyme compartment to the receptor compartment.

Acetylcholine mediation of the contractile response to histamine in human bladder detrusor muscle.

In Krebs solution, histamine evokes in human bladder detrusor muscle strips a dose-dependent contractile response which consists of two pharmacologically distinct responses: a high-sensitivity response evoked at 0.4-2 microM histamine, which is potentiated by neostigmine (0.1 microM) or blocked by atropine (0.1 microM) or ranitidine (1 microM); a low-sensitivity response evoked at 4-40 microM histamine and blocked by dimethindene or diphenhydramine. These findings suggest that the contractile response to low doses of histamine is mediated by acetylcholine released from a site proximal to the muscle. This effect of histamine seems to be mediated by a site which is insensitive to the H1 antagonists dimethindene and diphenhydramine but blocked by the H2 antagonist ranitidine.

Histamine-evoked acetylcholine release in sensitized tracheal preparation

The contractile response to histamine of tracheal muscle was studied in preparations from BSA-sensitized and non-sensitized guinea-pigs. Sensitization did not enhance the overall response to histamine. However, this response showed evidence of acetylcholine participation. In sensitized preparations, atropine (0.1 microM) caused a significant depression of the dose response to histamine (n = 11, p = 0.028), especially in the range 2-8 microM. Physostigmine (0.1 microM) significantly potentiated the effect of histamine (n = 8, p = 0.003), especially at greater than 4 microM histamine. The response to histamine of non-sensitized preparations was not altered by atropine (n = 11) or physostigmine (n = 8). The following agents did not discriminate between sensitized and non-sensitized preparations: Famotidine, an H2 antagonist; dimaprit, an H2 agonist; thioperamide, an H3 antagonist; alpha-methylhistamine, an H3 agonist; gallamine, an M2 antagonist, suggesting that muscarinic M2 receptor dysfunction alone is not sufficient to cause bronchial hyper-responsiveness. The results show that sensitization causes a change in the components of the contractile response to histamine rather than bronchial hyper-responsiveness to this agent.

Antihistaminic properties of AF-14, an experimental quinuclidine derivative: Discrimination between two histaminergic sites in both guinea-pig ileum and bladder

AF-14 (1-aza-4-phenyltricyclo[6.2.2.0(2,7)]dodecan-5-one), a selective antimuscarinic agent, was shown to block the histamine-induced contractile response in Krebs solution in guinea-pig ileum and bladder. Careful linear regression analyses of the concentration-response relationship revealed that AF-14 antagonism consisted of two sequential and distinct phases, corresponding to an early phase and a late phase of the contractile response to histamine. In the late phase, the action of AF-14 was consistent with competitive antagonism, its pA2 (95% confidence limits) being 5.80-6.06 (ileum) and 5.66-5.80 (bladder). In the early phase, AF-14 almost completely blocked the contractile response at a concentration of 100-300 nM, which was much less than required to block the late phase or cholinergic contractions of the ileum or bladder. It is concluded that AF-14 discriminates between two histamine-sensitive sites that mediate muscle contraction in each of the two organs.

Cholinoceptor Characterization Reconsidered in the Light of Diversity of Response

The current concern with central cholinergic hypofunction as a unique pathophysiological correlate of Alzheimer disease and related disabilities is perhaps behind the recent revival of interest in the pharmacology of the cholinoceptive site (1). Conceivably, and given the assumption that such sites are sufficiently dissimilar among different tissues, say brain and muscle, to allow a differential response to a cholinomimetic molecule, then the prospects of drug-based management of such disabilities could be set on a rational basis. Historically, the issue of possible heterogeneity among cholinoceptive sites of the so-called muscarinic type had a relatively early head-start when it was realized that such drugs as acetylcholine (ACh) and carbachol (CCh) were not proportionately active in the heart on one hand and the intestine or bladder on the other (2), thus suggesting some organ or tissue specificity in the respective molecule. The pharmacological history of the cholinoceptor does not lack other examples of tissue or rather effect specific agents (3–8). These, however, failed to evoke sufficient interest as potential probes for cholinoceptor characterization. The prevailing approach regarded antagonists as unequivocal probes, and their relative affinity to the cholinoceptor, expressed as the equilibrium dissociation or association constant, as an index of its identity. Thus, and for long, atropine or a related molecule served as a universal touchstone for the unfailing identification of the cholinoceptor. The popularity of these probes is due to a rare combination of high potency and high specificity. Yet, they could not discriminate among receptor subtypes; so much so, that leading investigators found no reason to assume the existence of cholinoceptor subtypes. The emergence of even more potent antagonists such as QNB which heralded the advent of the popular radioligand binding assay (9), opened additional avenues for cholinoceptor characterization, but then an almost dogmatic adherence to “affinity” remained the major criterion for defining cholinoceptor identity which remained uniform with only a few suggestions to the contrary (10, 11).