Changes of the responses of single sympathetic ganglionic neurones to substance P following desensitization.

1. The neuropeptide substance P (SP) exerts an excitatory effect on sympathetic neurones by inhibiting a time- and voltage-dependent potassium current. During prolonged application of SP, the response desensitizes. The changes in kinetics of the SP response in single neurones after desensitization have been studied in an attempt to gain some insight as to the molecular mechanism of desensitization in live, functioning neurones. 2. Desensitization to SP resulted in subsequent SP responses being smaller, but the time course was unchanged in desensitized cells compared with non-desensitized cells. 3. Experimental manipulations were performed to decrease receptor and G protein function for comparison to desensitization. Intracellular application of GDPbetaS, to decrease G protein function, led to successive responses to agonist becoming smaller and slower. When functional muscarinic receptors were decreased by extracellular application of propylbenzilylcholine mustard (PrBCM), the response to muscarine became smaller, but the time course was unchanged compared with the change in time course produced by PrBCM vehicle alone. 4. The results have also been compared with simulations from a mathematical model of drug-receptor-G protein interactions. Under a constrained set of conditions, the model predicts that decreasing the size of the G protein pool will decrease both the magnitude and the time course of the response to agonist. Decreasing receptor levels results in a more efficient decrease in the magnitude of the response but no change in the time course of the response. 5. These data provide evidence that desensitization of the response to SP in single neurones results from a decrease in functional receptors.

Protein kinase C mediates increase of Ca2+ sensitivity for contraction by cholinoceptor partial agonist in ileal longitudinal muscle of guinea pig.

1. Experiments were designed to study the roles of protein kinase C in carbachol- and pilocarpine-induced contraction and the increase in cytosolic Ca2+ concentration ([Ca2+]i) in guinea pig ileal longitudinal muscle. 2. The protein kinase C inhibitors, GF 109203X (10 microM), calphostin C (10 microM) and H-7 (10 microM), reduced the maximum of the concentration response curve produced by pilocarpine more effectively than that produced by carbachol. 3. The slopes of the regression lines between [Ca2+]i and tension development for pilocarpine and carbachol in tissues treated with GF 109203X were significantly gentler than those for untreated tissues. 4. The protein kinase C alpha- and beta 1 selective inhibitor Goe 6976 (1 microM) decreased both [Ca2+]i and contraction, but did not affect the slopes of the regression lines for pilocarpine and carbachol. 5. These results suggest that protein kinase C (both n- and/or a-type) plays an important role in the increase of Ca2+ sensitivity of the contractile element, and that pilocarpine mainly activates the protein kinase C-dependent pathways for contractile mechanisms in guinea pig ileal longitudinal muscle.

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.

Cardiac muscarinic receptors. Relationship between the G protein and multiple states of affinity.

An expanded version of the mobile receptor model has been assessed in studies on the binding of N-[3H]methylscopolamine and [35S]GTPgammaS to cardiac muscarinic receptors and their attendant G proteins in ventricular membranes from hamster. The model comprises two pools of receptor, one of which lacks G proteins, and a heterogeneous population of G proteins that compete for the receptor within the G protein-containing pool. To guide the formulation of the model itself and to define the various parameters, data were combined from assays performed under various conditions with native membranes and following irreversible blockade of about 80% of the receptors with propylbenzilylcholine mustard. Multiple G proteins are indicated primarily by multiple states of affinity evident in the dose-dependent effect of guanyl nucleotides on the binding of carbachol; G protein-free receptors are indicated by sites of low affinity for carbachol that survive treatment with the mustard. The expanded model generally succeeds where more frugal schemes have been inadequate, but it nevertheless fails to yield a mechanistically consistent description of the data. Guanyl nucleotides and partial alkylation do not affect the inhibitory potency of carbachol in a manner consistent with their supposed effect on the equilibrium between uncoupled and G protein-coupled receptors. As inferred from the model, G proteins are lost upon alkylation of the receptor, and their numbers are regulated by guanyl nucleotides. Parameters estimated via N-[3H]methylscopolamine are wholly inconsistent with the same parameters estimated via [35S]GTPgammaS. The failure of the model suggests that multiple states of affinity may not arise from a ligand-regulated equilibrium between free receptors and G proteins on the one hand and one or more RG complexes on the other.

Drug receptor mechanisms in smooth muscle: beta-chloroethylamine-sensitive and -resistant receptor mechanisms.

Both alpha1-adrenoceptors and M3-cholinoceptors can be divided into two subtypes discriminated by the beta-chloroethylamines, chloroethylclonidine and propylbenzilylcholine mustard (PrBCM), only in the presence of GTP. The full agonists interact with both subtypes to induce responses. The partial agonists activate one of them to induce responses but behave as competitive antagonists when they interact with the other. The responses mediated through the receptors that are activated by the partial agonists are resistant to myosin light chain kinase inhibitors, while the response through the activation of the other receptors are suppressed by the inhibitors. The receptor stimulations through alpha1A-adrenoceptor and PrBCM-sensitive M3-cholinoceptor subtypes mainly activate the myosin light chain-phosphorylation-independent pathway mediated through protein kinase C and low molecular weight GTP-binding protein, whereas the stimulations through alpha1B-adrenoceptors and the PrBCM-phosphorylation-dependent pathway are directly related to Ca2+/calmodulin.

Muscarinic receptor reserve for inhibition of cAMP accumulation in bovine trachealis cells.

In airway smooth muscle, muscarinic agonists inhibit synthesis of adenosine 3',5'-cyclic monophosphate (cAMP). The goal was to characterize the relationship between agonist occupancy of muscarinic receptors and regulation of cAMP for bovine trachealis cells. For intact cells dispersed by enzyme, carbachol maximally inhibited 58 +/- 4% (mean +/- SE, n = 5) of isoproterenol-stimulated cAMP accumulation at low concentrations [log half-maximal effective concentration (EC50) = -8.34 +/- 0.16]. In radioligand binding experiments, carbachol competed for [3H]quinuclidinyl benzilate (n = 7) and [N-methyl-3H]scopolamine (n = 3) binding sites on intact cells with both low (log KL = -4.26 +/- 0.06 and -4.50 +/- 0.20, respectively) and high affinities (log KH = -5.91 +/- 0.24 and -6.39 +/- 0.19, respectively). In separate experiments, a fraction of the muscarinic receptors on intact cells were inactivated with either phenoxybenzamine (POB) or propylbenzylcholine mustard (PBCM). We compared equally effective concentrations of carbachol before and after partial inactivation of receptors, and the calculated equilibrium dissociation constants for agonist (log KA = -4.36 +/- 0.42 to -3.20 +/- 0.40 for POB; log KA = -4.27 +/- 0.45 for PBCM) were much greater than the half-maximally effective concentration of carbachol in control cells (log EC50 = -8.34 +/- 0.16). Based on these equilibrium dissociation constants, we calculated that maximum responses (EC95) to carbachol were obtained by occupancy of 0.8% of the receptors coupled to cAMP regulation. We concluded that muscarinic inhibition of cAMP accumulation is characterized by a muscarinic receptor reserve.

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.

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.

Absence of receptor reserve at hippocampal muscarinic autoreceptors which inhibit stimulus-dependent acetylcholine release.

The extent of reserve among inhibitory muscarinic autoreceptors on hippocampal cholinergic nerve terminals was examined in superfused calcium-naive synaptosomes. The tissues were treated with the irreversible muscarinic cholinergic receptor antagonist propylbenzilycholine mustard (PrBCM) and then used to assess the functional status of autoreceptors through acetylcholine (ACh)-induced inhibition of calcium-evoked [3H]ACh release. PrBCM treatment caused a marked reduction in the density of high-affinity [3H]quinuclidinyl benzilate binding sites (46%, 72% and 90% reductions after 3, 6 or 10 nM PrBCM, respectively) but had no apparent influence on the binding affinities or relative proportions of high- and low-affinity binding sites for the M1-selective antagonist pirenzepine or the agonist ACh. In vehicle-treated tissues, ACh was a potent (EC50 = 240 nM) and efficacious (maximal inhibition of stimulated [3H]ACh release = 65%) agonist at muscarinic autoreceptors. However, after PrBCM treatment, the maximal inhibition for ACh was greatly attenuated (35% and 17% for 3 and 6 nM PrBCM, respectively) with no concurrent changes in the EC50 or slope factor. Comparisons of equieffective agonist concentrations before and after receptor occlusion revealed a direct linear relationship between autoreceptor occupancy and inhibition of [3H]ACh release with close agreement between the calculated agonist dissociation constant (KA = 220 nM) and the EC50 for ACh. Pretreatment with 100 nM atropine methylbromide completely prevented PrBCM-induced reductions in muscarinic cholinergic receptor binding and autoreceptor function. These results support the conclusion that muscarinic autoreceptors on hippocampal nerve endings exhibit little or no reserve for inhibition of ACh release by the endogenous neurotransmitter.

Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells.

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.

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.

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.

Differential effects of alkylating agents on the multiple muscarinic receptor subtypes linked to activation of phospholipase C by carbachol in rat brain cortical membranes.

Muscarinic cholinergic receptor function in rat brain cortex was characterized by performing binding assays with [3H](-)quinuclidinyl benzilate ([3H]QNB) in parallel with assays of phospholipase C (PLC) activation by carbachol using membrane preparations and exogenous [3H]-phosphatidylinositol 4,5-bisphosphate ([3H]PIP2). Competitive binding studies revealed high- and low-affinity binding sites for the receptor antagonists, pirenzepine, methoctramine and the p-fluoro analog of hexahydro-sila-difenidol (p-F-HHSiD). Carbachol-stimulated [3H]-phosphatidylinositol 4,5-biphosphate breakdown was specifically inhibited by pirenzepine and p-F-HHiSD. The inhibition curves for these antagonists were best described by interactions at two sites. There was quantitative agreement between the antagonist affinity constants and the proportion of high- and low-affinity sites derived in functional and binding studies. The characteristics of the putative subtypes of muscarinic receptors and their stimulation of phospholipase C was examined after treatment with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and propylbenzilylcholine mustard. Loss of receptors was closely correlated with loss of PLC activation by carbachol, without alteration of the EC50 value (21 microM) of this agonist, clearly demonstrating a lack of receptor reserve. When both alkylating treatments were adjusted to induce a decrease of 60% in the maximal number of [3H]QNB binding sites, a similar (60%) reduction in the maximal effect of carbachol on PLC activation was found. However, the characteristics of the remaining receptors after the treatment with the two alkylating agents differ markedly as determined by competition of pirenzepine, p-F-HHSiD and methoctramine for [3H]QNB binding, and for inhibition of carbachol-stimulated phospholipase C by pirenzepine and p-F-HHSiD.(ABSTRACT TRUNCATED AT 250 WORDS)

Propylbenzilylcholine mustard has greater specificity for muscarinic m2 receptors than for m3 receptors present in cerebellar granule cell culture from rat.

Both muscarinic m2 receptors that inhibit adenylyl cyclase and m3 receptors that stimulate the hydrolysis of inositol phospholipids are expressed in cerebellar granule cells. In order to determine whether a reserve population of either of these receptors is present in this cell culture, the irreversible muscarinic receptor antagonist, propylbenzilylcholine mustard (PBCM), was used at different concentrations to bind various proportions of available muscarinic receptors. After pretreating the cell cultures with low concentrations of PBCM (< 1 nM), the muscarinic m2 receptor-mediated response decreased. Higher concentrations of PBCM (1-3 nM) were needed to reduce the muscarinic m3 receptor-mediated response. These results suggested that either a reserve population of muscarinic m3 receptors is present or that PBCM shows greater specificity for muscarinic m2 receptors. Because the muscarinic m2 receptor is a 66 kDa protein, whereas the muscarinic m3 receptor is a 92 kDa protein, these receptors can be separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after being labeled with [3H]PBCM. PBCM appears to specifically bind the 66 kDa m2 receptor at concentrations lower than those required to bind to the 92 kDa m3 receptor. A linear correlation was found between the increased binding of [3H]PBCM to each receptor and the proportional loss of that receptor-mediated response. Thus, a reserve population of either muscarinic m2 or m3 receptors does not appear to exist in cerebellar granule cells. These studies also show that PBCM has greater affinity for the muscarinic m2 receptor than the muscarinic m3 receptor.

Characterization of subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle.

The subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle was examined using four selective muscarinic antagonists, pirenzepine, AF-DX 116, himbacine and 4-DAMP. The pA2 values of the four antagonists against pilocarpine were not different from their values against carbachol after the treatment with PrBCM and was identified with the values for the m3 subtype. These results suggest that the subtype of PrBCM-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle is the m3 subtype only and not other subtypes.

[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.

Propylbenzilycholine mustard (PrBCM)-sensitive cholinoceptors and contractile response to partial agonist in guinea pig ileal muscle.

Pilocarpine, a partial agonist, activates propylbenzilylcholine mustard (PrBCM)-sensitive cholinoceptors in the guinea pig ileal longitudinal muscle, while carbachol, a full agonist, predominantly activates PrBCM-resistant ones. Carbachol behaves as a partial agonist in the preparation treated with phenoxybenzamine and mainly activates PrBCM-sensitive cholinoceptors, as phenoxybenzamine preferably blocks PrBCM-resistant ones. The receptor occupancy-response curve for carbachol became a rectangular hyperbola, while pilocarpine showed a linear relation. After occlusion of cholinoceptors with phenoxybenzamine, carbachol showed a linear receptor occupancy-response relation, suggesting that its contraction mechanisms after occlusion of cholinoceptors resemble those for pilocarpine. Both the agonists induced an increase in cytosolic Ca2+ concentration [( Ca2+]i) and tension development in a concentration-dependent manner under the conditions used herein. The slopes of the regression lines between [Ca2+]i and tension development for pilocarpine in the untreated preparation and for carbachol in the preparation treated with phenoxybenzamine were significantly steeper than that for carbachol in the untreated preparation, suggesting that carbachol in the phenoxybenzamine-treated preparation and pilocarpine induced a greater tension for a given increase in low [Ca2+]i than did carbachol. Thus an activation of PrBCM-sensitive cholinoceptors might enhance the Ca2+-sensitivity of the contractile elements.

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.

Effect of temperature reduction on responsiveness to cholinomimetics in the taenia caeci of the guinea-pig.

1. The effect of temperature reduction on the interaction of carbachol (CCh) and McN-A-343 (McN) with muscarinic receptors in the guinea-pig taenia caeci was investigated. 2. McN, a partial agonist, acted on the smooth muscle to produce contraction. The response was unaffected by tetrodotoxin and the pKB for inhibition by pirenzepine was 6.8, indicating that ganglionic M1 receptors were not involved in the response. 3. Reduction in temperature from 37 degrees C to 18 degrees C for 3 h led to a marked reduction in the contractile response to McN (2-200 microM) but no reduction in the response to CCh (0.1-3 microM). 4. The reduction in temperature was not accompanied by any change in the affinity of McN or CCh for muscarine receptors in binding experiments with [3H]-QNB. 5. The KA value for CCh determined after irreversible receptor inactivation with propylbenzilylcholine mustard followed by ca 60-min wash-out was 7.6 microM, a value similar to that obtained in binding experiments. 6. The EC50 for McN in producing contraction at 37 degrees C (2.1 microM) was similar to the KA value for the partial agonist obtained in experiments with the irreversible antagonist phenoxybenzamine (2.5 microM). It was also similar to the KB value determined at 18 degrees C (3.4 microM) when McN could be used as an antagonist of contractions to CCh. 7. At 18 degrees C, phosphatidylinositol (PI) hydrolysis by CCh was reduced to 23% of that at 37 degrees C. 8. It is concluded that reduction of muscarinic receptor activation of the PI pathway by cholinomimetics with lowering of the temperature could account for the findings with McN on contractility.