Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: requirement of endogenous acetylcholine for outgrowth initiation.

To better understand the mechanisms of skin re-epithelization, we developed a simple technique that assays the outgrowth of human keratinocytes. Second-passage foreskin keratinocytes were inoculated at high cell density into 3-mm wells cut from agarose gels in standard 6-well tissue culture dishes. The cells settled on the dish bottom and formed a confluent colony. The cells at the periphery of the colony flattened, spread their cytoplasm, and moved away over the dish surface under the agarose gel. The morphology of migrating keratinocytes was observed microscopically through the transparent agarose, and the migration distance was measured after the gels were removed and after cells were fixed and stained. To determine which cell activities were involved in the outgrowth, the effects of cholinergic compounds on keratinocyte outgrowth were compared with their effects on keratinocyte proliferation, cell-plastic attachment, and spreading measured in separate sets of experiments. Outgrowth was inhibited by the specific inhibitor of acetylcholine synthesis bromoacetylcholine (0.05 mM) and restored by 5 mM exogenous acetylcholine. The irreversible muscarinic antagonist propylbenzilylcholine mustard (0.05 mM) abolished the restorative effects of exogenous acetylcholine, and also inhibited outgrowth of intact keratinocytes. In keratinocyte cell cultures, bromoacetylcholine stopped cell division. Propylbenzilylcholine mustard increased cell number, but interfered with cell-plastic attachment and spreading. This suggests that cell-matrix attachment, spreading, and locomotion of human keratinocytes, but not mitosis, mediate the earliest stages of skin re-epithelization, and that endogenous acetylcholine regulates these keratinocyte functions. Specifically, keratinocyte acetylcholine is required to initiate outgrowth.

Human muscarinic receptors expressed in A9L and CHO cells: activation by full and partial agonists.

1. A comparative study of receptor activation by ten full and partial muscarinic agonists was undertaken on the five subtypes of human muscarinic receptors expressed at similar receptor densities in Chinese hamster ovary (CHO-K1) cells. In addition, m1, m2 and m3 receptors were expressed in mouse fibroblast A9L cells in order to compare the influences of cell type on agonist activation of these receptors. 2. Receptor-effector coupling efficiencies were greater in CHO than A9L cells and agonists displayed greater potencies and similar or greater intrinsic activities at CHOm1 and CHOm3 than A9Lm1 and A9Lm3 receptors. Although m2 receptor density was 6 fold higher in A9L than CHO cells, carbachol elicited significantly greater inhibition of adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in CHOm2 cells. These data suggest that not only receptor density but receptor-effector coupling and/or coupling efficiencies play significant roles in agonist-induced responses. 3. In CHO cells, receptor-effector coupling efficiencies were m3 = m1 > m5. Although CHOm5 receptors were the least efficiently coupled, some partial agonists displayed higher intrinsic efficacies at m5 than m3 receptors suggesting that, in CHO cells, m5 and m3 receptors may activate different G proteins and/or effectors to stimulate inositol monophosphate (IP1) formation. 4. McN-A-343 was a functionally selective m4 agonist. It had little or no agonist activity at m3 receptors expressed in either A9L or CHO cells. The slopes of McN-A-343 concentration-response curves inCHOm2 cells were significantly lower than the slopes obtained with this compound in CHOm4 cells suggesting that the mode of activation by McN-A-343 differed between the two muscarinic receptors negatively coupled to adenylyl cyclase.5. Cloned receptors provide valuable tools for the study of agonist-receptor interaction and agonist receptor activation but caution should be applied in assuming that the results are valid for all cell types or for tissue-expressed receptors.

Ligand binding to muscarinic receptors in intact longitudinal muscle strips from guinea-pig intestine.

1 The binding of ligands to muscarinic receptors in intact longitudinal muscle strips from guinea-pig small intestine has been determined by measuring the inhibition of the irreversible binding of [3H]-propylbenzilylcholine mustard ([3H]-PrBCM). 2 The IC50 values for inhibition of [3H]-PrBCM binding by a given ligand were generally higher in intact strips than those reported for broken-cell preparations. This effect is probably due, at least in part, to the presence of an access-limitation factor in the kinetics of the irreversible binding of [3H]-PrBCM to the intact tissue. 3 The mean Hill coefficients for antagonist binding approached unity, but those for strong agonists were significantly less than unity. There was, with the possible exceptions of hexyltrimethylammonium and oxotremorine, reasonably good agreement with the Hill coefficients reported for brain homogenates.

Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea-pig taenia caeci.

1. The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR-602, and McN-A-343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea-pig caecum. 2. Compared to CCh, ACh was a full agonist for contraction but AHR-602 and McN-A-343 were partial agonists producing 80-85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR-602 and McN-A-343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh. 3. CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mM) was inhibited by atropine (IC50 8.5 nM) and pirenzepine (IC50 450 nM). The accumulation of inositol phosphates in the presence of AHR-602 or McN-A-343 was not significantly different (P greater than 0.05) from basal levels. 4. A concentration of 0.2 mM AHR-602 produced a parallel shift of the concentration-response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mM) was greater than 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN-A-343 (20 microM) produced a flattening of the concentration-response curve to CCh for inositol phosphate accumulation. 5. The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2-muscarinic receptor. AHR-602 and McN-A-343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.

Propylbenzilylcholine mustard is selective for rat heart muscarinic receptors having a low affinity for agonists.

Propylbenzilylcholine mustard (PrBCM), an irreversible muscarinic antagonist, inactivated receptors with a low affinity for agonists faster than those with a high affinity in rat heart membranes. This result was obtained using either: (a) a low ionic strength buffer (allowing heterogeneity among antagonist binding sites, (b) the same buffer enriched with GTP, or (c) a high ionic strength buffer (where antagonists showed similar binding characteristics to all receptors). These data suggest either that PrBCM is a 'selective' antagonist which detects conformational differences between low and high affinity receptors, or that the agonist affinity of cardiac muscarinic receptors is determined, in part, by the relative concentrations of receptor and GTP binding protein.

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca(2+)-mobilization and Ca(2+)-influx pathways in taenia caeci.

1. We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN-A-343 and AHR-602 with those of carbachol in smooth muscle of guinea-pig taenia caeci. 2. McN-A-343 and AHR-602, as well as carbachol, completely displaced the atropine-sensitive binding of [3H]-quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol > McN-A-343 >> AHR-602. 3. In the presence of 2.2 mM extracellular Ca2+, McN-A-343 and AHR-602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4-DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF-DX 116 (M2 selective). These contractions were inhibited by an L-type Ca2+ channel blocker, verapamil. 4. In Ca(2+)-free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN-A-343 and AHR-602. Application of McN-A-343 or AHR-602 inhibited the carbachol-induced contraction in Ca(2+)-free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5. The EC50 value for carbachol-induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6. The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor-mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN-A-343 and AHR-602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.

Affinity and efficacy of racemic, (+)-, and (-)-methacholine in muscarinic inhibition of [3H]-noradrenaline release.

The right postganglionic sympathetic nerves of rat isolated perfused hearts (previously loaded with [3H]-noradrenaline) were stimulated electrically with 10 trains of 10 pulses at 10 Hz. The inhibition by methacholine of stimulation-evoked [3H]-noradrenaline overflow into the perfusate (determined in the presence of corticosterone, desipramine, phentolamine, and propranolol) was taken as a measure for activation of presynaptic muscarinic receptors. The evoked [3H]-noradrenaline overflow was inhibited by (+)-, racemic, and (-)-methacholine in a reversible and concentration-dependent manner. The concentration causing 50% inhibition (IC50) was 0.1, 0.26, and 65 microM, respectively, resulting in an isomeric potency ratio IC50 (+)/IC50(-) of 650. The dissociation constant KA of the (+/-)- or (+)-methacholine-presynaptic receptor complex was determined after fractional receptor inactivation according to Furchgott & Bursztyn (1967) with phenoxybenzamine or propylbenzilylcholine mustard as irreversible antagonists of muscarinic receptors. KA for (-)-methacholine was estimated according to Mackay (1966). KA of (+)-, (+/-)-, and (-)-methacholine were 2.5, 4 and 440 microM, resulting in an isomeric affinity ratio KA (+)/KA(-) of 180. The discrepancy between the isomeric IC50 ratio and the isomeric KA ratio is explained by a higher intrinsic efficacy of the (+)-enantiomer compared to the (-)-enantiomer. Thus, (+)-methacholine has to occupy fewer receptors to induce a given inhibition of release than its antipode as revealed by a plot of fractional receptor occupancy vs response. The results show that, in the effector system of presynaptic muscarinic inhibition, methacholine enantiomers differ greatly not only in affinity for the receptor, but also to some extent in the efficiency of signal transmission, and both parameters contribute to the high isomeric potency ratio. The activity of the racemate is fully accounted for by the activity of the (+)-enantiomer.

Effects of propylbenzilylcholine mustard on injection into the liquor space of cats.

In unanaesthetized cats the effects were examined of propylbenzilylcholine mustard (PrBCM) on injection into the cannulated cerebral ventricles and cisterna magna. Extreme motor excitation, vocalization, shivering leading to fever, tachypnoea, panting, piloerection and salivation were produced on ventricular, vigorous scratching bouts on cisternal, injections. The sites of these actions are discussed. None of the effects was produced by atropine similarly injected. All effects were suppressed by anaesthetizing doses of pentobarbitone sodium injected intraperitoneally.

Blockade of cholinergic receptors by an irreversible antagonist, propylbenzilylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning.

Studies were made on the effects of blockade of muscarinic acetylcholine (mACh) receptors in the rat cerebral cortex on learning and memory assessed by performance of a step-through passive avoidance task. Bilateral injection of propylbenzilylcholine mustard (PrBCM) into both the frontal and parietal cortex at doses of 2.25 X 4 to 22.5 X 4 micrograms decreased mACh receptors dose-dependently, as assessed by [3H]quinuclidinyl benzilate binding studies. When the training trial of a step-through passive avoidance task was performed 24 h after injection of 7.5 X 4 to 22.5 X 4 micrograms PrBCM into the frontal and parietal cortex, and then a retention test was made 24 h after the training trial, the treated rats showed shorter latencies than controls. In contrast, injection of PrBCM into the occipital cortex had no significant effect on performance in the test. These results confirm the notion that cholinergic neurotransmission in the cerebral cortex, especially the frontoparietal cortex, is important in learning and memory. The effects of injection of PrBCM (22.5 X 4 micrograms) into the frontoparietal cortex on 3 postulated phases of the learning and memory process (i.e. registration, retention and recall) were also examined. When PrBCM was injected 24 h before the training trial, no retention of the task was observed 14 days after the training trial. However, when PrBCM was injected 24 h after the training trial, retention of the task 14 days after the training trial was not affected. When PrBCM was injected 3-24 h after the initial training trial, the latencies in the retention test examined 24 h later were shorter than those of control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac M2 receptors consist of two different types, both regulated by GTP.

Cardiac muscarinic receptors are predominantly M2 receptors, and have three agonist binding sites (super-high(SH), high(H) and low(L) affinity agonist binding sites). Treatment of cardiac membranes with 50 nM propylbenzilyl choline mustard (PrBCM) caused 88% loss of binding sites for [3H]QNB. Carbamyl choline (CCh) inhibits this alkylation dose dependently and, theoretically, generates uneven alkylation of multiple agonist binding sites. Pretreatment of the membranes with 50 nM PrBCM and 0.5 mM CCh resulted in almost complete disappearance of L sites with similar degrees of conservation of H sites and SH sites. In these pretreated membranes, guanine nucleotide and sulfhydryl reagent caused a change in the ratio of residual SH and H sites but not of L sites though previous studies showed that, in intact membranes, these reagents affected the ratio of SH and L sites without significantly changing that of the H site. These results indicate the existence of two equilibria regulated by guanine nucleotide and sulfhydryl reagent in cardiac muscarinic receptors: one between SH and H sites and the other between H and L sites. The participation of GTP binding protein(s) in all cardiac muscarinic responses is suggested.

The SH-H subgroup of cardiac M2 receptors (M2 alpha) inhibits adenylate cyclase activity.

Muscarinic receptors in the guinea-pig heart seem to consist entirely of M2 receptors, but are coupled with several responses including inhibition of adenylate cyclase activity. On the other hand three affinity states (SH, H and L) can be distinguished in cardiac membranes with muscarinic agonists such as carbachol. We showed previously that the three agonist binding states were the sum of two equilibria (SH-H and H-L subgroup), both regulated by GTP-binding protein(s). In this study we determined which subgroup was responsible for the inhibitory effect of muscarinic M2 receptors on adenylate cyclase activity. The ED50 values for this response of four muscarinic agonists, acetylcholine, carbachol, pilocarpine and oxotremorine corresponded with the binding KD values of H (acetylcholine and carbachol) and LO/P (pilocarpine and oxotremorine) sites. After alkylation of spare receptors, the ED50 value of carbachol was changed from 4.3 to 5.6 microM, which corresponded with the KD value of the H site. Furthermore, the four agonists were almost fully active when membrane preparations were pretreated with propylbenzilylcholine mustard (PrBCM) in the presence of carbachol to destroy the H-L subgroup, whereas after pretreatment with PrBCM and atropine, which alkylated both types of subgroups evenly, the decrease in the number of receptors was proportional to the decrease in the inhibitory effect on adenylate cyclase activity. These results suggest that only the SH-H subgroup (M2 alpha) is responsible for the inhibitory action of muscarinic receptors on adenylate cyclase activity in the heart.

Muscarinic M2 receptor synthesis: study of receptor turnover with propylbenzilylcholine mustard.

We have investigated the rate and the functional responsiveness of the newly synthesised M2 muscarinic receptors in HEL 299 cells following propylbenzilylcholine mustard treatment at 37 degrees C. Propylbenzilylcholine mustard induced a dose-dependent loss of the hydrophilic ligand [3H]N-methylscopolamine binding sites with 80% inactivation at 0.1 microM. The rate of muscarinic receptor synthesis in these cells, estimated from wash-out experiments following propylbenzilylcholine mustard treatment, was very slow and returned to control values after 36 h of propylbenzilylcholine mustard removal. The recovery of muscarinic receptors was blocked by the cycloheximide pre-treatment, indicating the synthetic pathway for the new receptors. In control cells as well as in cells treated with propylbenzilylcholine mustard and allowed to recover for 12 h, carbachol still inhibited forskolin-induced cAMP accumulation. These results show that (i) the rate of M2 muscarinic receptor synthesis is slow (ii) the recovery of receptors is mainly through increased synthesis and (iii) the newly synthesised receptors retain their full functional activity.

Activation of propylbenzilylcholine mustard-sensitive muscarinic cholinoceptors more effectively utilizes cytosolic Ca2+ for contraction in guinea-pig intestinal smooth muscle.

A 50-min treatment of longitudinal smooth muscle of guinea-pig ileum with propylbenzilylcholine mustard (PrBCM, 3 x 10(-6) M) irreversibly inhibited the responses elicited by carbachol. However, a 90-min treatment with PrBCM had no further significant inhibitory effect on the responses to carbachol, suggesting that there are two subtypes of muscarinic cholinoceptors, PrBCM-sensitive and PrBCM-insensitive receptors. Carbachol caused a rapid increase in cytosolic Ca2+ concentrations ([Ca2+]i), which was followed by a rapid increase in muscle tension in both untreated and PrBCM-treated preparations. There was a positive correlation between [Ca2+]i (R340/380) and tension developed in response to carbachol. A regression line for the two responses was obtained in each preparation. The slope of the line obtained with untreated preparations was steeper than that obtained with PrBCM-treated preparations. These data suggest that, upon activation, PrBCM-sensitive receptors use cytosolic Ca2+ more effectively than PrBCM-insensitive receptors.

The effect of propylbenzilylcholine mustard on contraction and radioligand binding parameters of muscarinic receptors in guinea pig ileum.

The receptor occupancy-biological effect relationship for muscarinic receptors in guinea pig ileal smooth muscle has been studied by comparison of radioligand binding and contractile response. Muscarinic receptors in homogenates of ileal smooth muscle were labeled with [3H]-1-Quinuclidinyl benzilate. Treatment with propylbenzilylcholine mustard (PrBCM), to inactivate irreversibly muscarinic receptors, caused a large dose dependent rightward shift of the dose-response curve to three agonistic furtrethonium derivatives with a concomitant decrease in maximal response. Using those data, the fraction of receptors remaining unoccupied (q-values) and "true affinity constants" (-log KA-values) were calculated. Exposure to 20 or 60 nM PrBCM for 15 minutes resulted in a 39% and a 61% reduction in specific [3H]-1-Quinuclidinyl benzilate binding sites respectively to be compared with a 62% and a 85% decrease expected from calculated q-values. KA-values for the methyl and ethyl derivative agreed well with the dissociation constants for the high affinity agonist sites determined from displacement of [3H-]-1-Quinuclidinyl benzilate. The KA-value for the propylfurtrethonium corresponds to the low affinity agonist dissociation constant. The fraction of receptors in the high affinity agonist state differs considerably for the three furtrethonium derivatives investigated. Neither the fraction of receptors in the high affinity agonist state, nor the ratio of dissociation constants for these states is affected by the alkylation of 85% of the functional muscarinic receptors. The inactivation of components of the effector system by PrBCM seems unlikely.

[Transition of drug receptor mechanisms].

It is generally accepted that the agonists, full agonist and partial agonist, interact with the same receptors according to the classical receptor mechanisms. We tried to modify the drug receptor mechanisms in muscarinic cholinoceptors, alpha 1-adrenoceptors and beta-adrenoceptors. In the muscarinic cholinoceptor, there are two subtypes of M3-cholinoceptors, propylbenzilylcholine mustard (PrBCM)-sensitive receptors and PrBCM-resistant ones. The full agonists contract the longitudinal muscle through the interaction of two cholinoceptors, PrBCM-sensitive and-resistant ones, while the partial agonists produce the contraction through only the activation of PrBCM-sensitive ones. Upon activation PrBCM-sensitive receptors may use cytosolic Ca2+ more effectively than PrBCM-resistant receptors. In the alpha 1-adrenoceptor, the full agonist induces contraction through both alpha 1A and alpha 1B subtypes and the partial agonist through only alpha 1A subtype. The adrenoceptors activated by full agonist may be partly different from that by partial agonist in the arteries. In both the common iliac artery and thoracic aorta treated with the irreversible antagonist, phenoxybenzamine the slopes of schild plots of the results obtained from an antagonism between full agonist (phenylephrine) and alpha 1A-selective competitive antagonist (WB4101) equal to 1, suggesting that phenoxybenzamine preferably interacts with alpha 1B subtype. In the beta-adrenoceptor, the pD2-values of the partial agonists obtained from the concentration-response curves are significantly different from their pA2-values against full agonist (isoprenaline). The Scatchard plot of the specific [3H]befunolol (the partial agonist) binding showed two affinity sites of the receptors in the absence of Gpp(NH)p but the low affinity site was reduced while the high affinity site was not affected in the presence of Gpp(NH) p. The beta-adrenergic partial agonists are able to discriminate these two different binding sites of the beta-adrenoceptors. Our results suggest that the receptors activated by full agonists are partly different from those by partial agonists in muscarinic cholinoceptors, alpha 1- and beta-adrenoceptors, and that the irreversible antagonist can discriminate between the sites interact with full agonists and those with partial agonists in muscarinic cholinoceptors and alpha 1-adrenoceptors.

Blockade of ACh receptors by PrBCM causes deficits in shuttle avoidance performance.

Studies were made examining the effect of blockade of muscarinic acetylcholine (mACh) receptors in the cerebral cortex of rats on their shuttle avoidance after training. Rats were given a session of shuttle avoidance tests once a day for 12 days. Then the irreversible antagonist of mACh receptors, propylbenzilylcholine mustard (PrBCM), was injected bilaterally into the cerebral cortex of rats showing avoidance rates of more than 75% in the last session, and avoidance rates were examined 24 hr later. The avoidance rates of the rats treated with 100 micrograms PrBCM were lower than those in the last session before treatment. The amount of mACh receptors in the cerebral cortex was decreased by PrBCM treatment, as shown by [3H]quinuclidinyl benzilate (QNB) binding studies performed just after measurement of the avoidance response. The present study indicates that cholinergic neurotransmission in rat cerebral cortex is involved in performing a learned shuttle avoidance.

Structural requirements for muscarinic receptor occupation and receptor activation by oxotremorine analogs in the guinea-pig ileum.

The muscarinic activities in the isolated guinea-pig ileum of nine analogs of oxotremorine, modified only in the amino group, were resolved into affinity and efficacy components. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. The dissociation constants (KA) thus obtained for oxotremorine (6.79 X 10(-7) M), oxotremorine methiodide (6.74 X 10(-6) M) and oxotremorine-M (2.92 X 10(-6) M) agreed well with their reported low-affinity dissociation constants (KL) determined in receptor binding studies. There was no correlation between relative affinities and efficacies of the nine agonists studied, suggesting different structural requirements for occupation and activation of muscarinic receptors in the guinea-pig ileum. Although oxotremorine had higher affinity than its analogs, some of the latter had substantially greater efficacy than oxotremorine. Thus, replacement of the pyrrolidine ring of oxotremorine by azetidino, dimethylamino or trimethylammonium groups was accompanied by a 4- to 7-fold increase in efficacy. A diethylamino group in place of pyrrolidine gave an 18-fold decrease in efficacy and a triethylammonium group abolished efficacy. The relative efficacies of the nine agonists were inversely correlated with the size of the amino or ammonium group. No significant correlation was observed between relative affinities for the receptor and size of the cationic head.

Diminished functional activity of newly synthesized muscarinic acetylcholine receptors in stably transfected Y1 adrenal cells.

We have examined the functional responsiveness of newly synthesized m2 muscarinic acetylcholine receptors in stably transfected Y1 adrenal cells. After inactivation of preexisting receptors with the covalent alkylating antagonist propylbenzilylcholine mustard, the number of cell surface receptors returned to control values over a 3-h period. After a 3-h recovery, the cells exhibited diminished sensitivity for muscarinic receptor-mediated inhibition of adenylyl cyclase activity, with much higher concentrations of agonist being required to elicit a response. The functional sensitivity returned to control values over a 12-18-h period. The decreased functional activity was not due to a decreased affinity of the newly synthesized receptors for agonist or to a decrease in the levels of inhibitory G proteins in the cells. The results suggest that muscarinic receptors may be synthesized in a form with diminished functional activity. The ability to study the maturation of receptor function in a transfected cell system should allow a combination of biochemical and molecular genetic approaches to analyze the synthesis and functional responsiveness of muscarinic receptors.

A difference in receptor mechanisms for muscarinic full and partial agonists.

Concentration-response curves of 4 muscarinic full agonists were progressively inhibited by 10 to 50-min treatments of the longitudinal muscle of guinea pig ileum with propylbenzilylcholine mustard (PrBCM, 3 x 10(-6) M). A 90-min treatment with PrBCM had no further significant inhibitory effect on their curves. The 50-min treatment with PrBCM (3 x 10(-6) M) completely inhibited the concentration-response curves of 6 partial agonists. The limiting effect of PrBCM observed on the concentration-response curves of the full agonists was not found on the curves of the partial agonists. These results suggest that there are two subtypes of M3-cholinoceptors, PrBCM-sensitive receptors and PrBCM-resistant ones. Pilocarpine, a partial agonist, shifted the concentration-response curve of carbachol, a full agonist, in a parallel fashion in the strips treated with PrBCM (3 x 10(-6) M) for 50 min, suggesting that an interaction of pilocarpine with PrBCM-resistant cholinoceptors does not induce contraction. The full agonists contract the longitudinal muscle through the interaction of two cholinoceptors, PrBCM-sensitive and -resistant ones, while the partial agonists produce the contraction through the activation of PrBCM-sensitive ones.

Differences in muscarinic receptor reserve for inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in chick heart cells.

Carbachol is 100 times more potent for inhibiting cyclic AMP formation than for stimulating phosphoinositide (PI) hydrolysis in chick heart cells. To determine whether this reflects differences in agonist affinity of the receptor(s) coupled to the two responses, we measured these functional responses following removal of receptor reserve with propylbenzilycholine mustard (PrBCM). Conditions of PrBCM treatment that led to progressive loss of up to 95% of the [3H]-N-methylscopolamine-binding sites decreased the potency but not the maximal capacity of carbachol to inhibit cyclic AMP formation. In contrast, there was a marked decrease in the maximal PI response to carbachol. The KA for carbachol, calculated by measuring functional responses following receptor inactivation, was similar whether the cyclic AMP or the PI response was examined. These KA values (approximately 40 microM) were similar to the KD calculated by examining carbachol competition for [3H]-N-methylscopolamine-binding sites on the intact cell. PrBCM treatment also decreased the maximal effect of oxotremorine on cyclic AMP formation under conditions in which carbachol remained a full agonist for this response. We interpret our data as indicating that: there is much greater receptor reserve in the coupling of muscarinic receptors to adenylate cyclase than to PI hydrolysis; this, rather than differences in receptor affinity underlies the disparate dose-response relationships for the two responses; and differences in the effects of weak agonist on the two responses may also reflect differences in receptor reserve. We suggest that muscarinic receptors with the same affinity for carbachol interact with different efficiency with the transducers (Gi and Gx) that regulate adenylate cyclase and phospholipase C.