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We used gene targeting to generate mice lacking the M1 muscarinic acetylcholine receptor. These mice exhibit a decreased susceptibility to pilocarpine-induced seizures, loss of regulation of M-current potassium channel activity and of a specific calcium channel pathway in sympathetic neurons, a loss of the positive chronotropic and inotropic responses to the novel muscarinic agonist McN-A-343, and impaired learning in a hippocampal-dependent test of spatial memory.

We investigated the effects of muscarinic acetylcholine receptor stimulation on the expression levels of the G-protein-coupled inwardly rectifying K+ channel (GIRK) subunits using solution hybridization and immunoblot analyses. We report here that treatment of chick embryos in ovo with muscarinic agonist causes decreases in mRNA levels encoding GIRK1 and GIRK4 in atria but does not alter GIRK1 expression in ventricles. In addition, GIRK1 protein levels also demonstrate a decrease in atria upon muscarinic acetylcholine receptor stimulation. Numerous receptors couple to the activation of the GIRK family of inwardly rectifying K+ channels; thus, these decreases represent a novel mechanism for regulating physiological responses to chronic agonist exposure.

Incubation of cultured embryonic chicken heart cells with transforming growth factor beta 1 (TGF-beta 1) resulted in a concentration- and time-dependent decrease in the number of muscarinic acetylcholine receptors (mAChR), which reached a maximum by 24 hr. Twenty-four hours following TGF-beta 1 treatment, cm2 and cm4 mAChR protein levels were decreased 59 and 41%, respectively, and cm2 mRNA and cm4 mRNA levels were both decreased by approximately 40%. Chick heart cells treated with TGF-beta 1 exhibited a decreased sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity compared with control cells. Thus, TGF-beta 1 stimulation of chick heart cells results in decreases in the expression of both cm2 and cm4 mAChR and in muscarinic responsiveness.

Treatment of chicken embryos in ovo for 8 hr with the muscarinic agonist carbachol results in an 85% reduction in the number of muscarinic acetylcholine receptors ( mAcChoR ) present in atrial membrane homogenates. Subsequent treatment of embryos with the muscarinic antagonist atropine results in a gradual increase in mAcChoR number, which returns to control levels after 14 hr. This recovery of receptor number is blocked by administration of the protein synthesis inhibitor cycloheximide, consistent with previous results in cell culture, which suggested that de novo protein synthesis is required for the recovery of mAcChoR after agonist-induced decreases. Measurements of the negative chronotropic response to applied carbachol with isolated atria show that even after recovery of receptor number to control levels the response to agonist is diminished. The IC50 for carbachol is shifted approximately equal to 10-fold from controls at 20 hr after atropine treatment, but less than 3-fold at 28 hr, with no further change in receptor number occurring over this time. This increase in physiological sensitivity is not blocked by cycloheximide. Receptors at 20 hr have binding constants for agonists and antagonists that are indistinguishable from controls. This implies that there is a defect in coupling of mAcChoR binding to the physiological response when mAcChoR reappear following agonist-mediated decreases in receptor number. Recovery of the ability of mAcChoR to inhibit adenylate cyclase parallels recovery of the beating response--that is, the IC50 is shifted approximately equal to 11-fold from controls at 20 hr after atropine treatment, yet only 3-fold at 28 hr. Thus, newly synthesized mAcChoR exhibit decreased physiological and biochemical responses to muscarinic agonists, suggesting that mAcChoR are initially synthesized in a less active form.

Previous work has demonstrated that prolonged treatment of chick embryos in vivo with muscarinic agonists leads to a decrease in the number of muscarinic acetylcholine binding sites in the heart and a decrease in the sensitivity of isolated atria to the negative chronotropic response to muscarinic stimulation. We show here that treatment with the agonist carbachol leads to greater decreases in receptor number than does treatment with oxotremorine. Simultaneous treatment with oxotremorine and carbachol resulted in partial blockage in the maximal decrease in receptor number seen after treatment with carbachol alone. After treatment of embryos in vivo with carbachol to decrease the reserve of spare receptors in the atria, oxotremorine could no longer elicit a significant negative chronotropic response under conditions in which carbachol was still effective. These results demonstrate that oxotremorine is a partial agonist at the muscarinic acetylcholine receptor in the embryonic chick heart.

A quantitative immunoblot assay was developed by using affinity-purified monospecific antibodies to quantitate levels of guanine nucleotide binding regulatory protein (G-protein) subunits in atria and ventricles during embryonic chicken cardiac development. The muscarinic acetylcholine receptor (mAChR) number was measured with [3H]quinuclidinyl benzilate. On day 10 of embryonic development (day 10E) there was no difference between the atrial and ventricular membrane concentrations of beta-subunit, G0 alpha subunit, or mAChR. The level of Gi alpha was found to be 44% greater in atria than in ventricles on day 10E. The atrial membrane concentration of beta-subunit increased 80% between day 13E and 15E, G0 alpha increased 46% between day 10E and 15E, mAChR increased 61% between day 10E and 12E, and Gi alpha decreased 34% between day 10E and 13E. The atrial levels of beta-subunit, G0 alpha, Gi alpha, and mAChR did not change further through day 20E. The ventricular membrane concentration of these proteins did not change between day 10E and 20E, except for that of G0 alpha, which increased 47% between day 15E and 20E. The atrial specific increase in beta-subunit correlated with a loss of GTP inhibition of basal adenylate cyclase activity. The difference in Gi alpha levels between atria and ventricles on day 10E correlated with a difference in carbachol sensitivity of atrial and ventricular basal adenylate cyclase activity. Thus, the levels of several components of the cholinergic neuroeffector pathway are regulated in a tissue-specific manner at a time that coincides with the onset of functional parasympathetic innervation of the embryonic chicken heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure of cultured embryonic chicken cardiac cells to the muscarinic agonist carbachol results in a 70-80% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed on the surface of the cells. Removal of the agonist results in a gradual increase in mAChR number because of the accumulation of newly synthesized receptors, reaching the control level in 14 hr. Measurements of increases in K+ permeability elicited by carbachol show that even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The EC50 for carbachol is 13-fold higher in cells that have been exposed to carbachol and allowed to recover for 18 hr than in control cells, but is not significantly different from the EC50 for control cells 24 hr after agonist removal. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at 18 hr, and recovers by 24 hr. These increases in sensitivity of mAChR-mediated responses are not blocked by administration of cycloheximide, and thus do not require de novo protein synthesis. The number of surface mAChR available for ligand binding can be reduced by 85-100% by treatment with the affinity-alkylating antagonist propylbenzilylcholine mustard. Newly synthesized mAChR that appear following affinity alkylation are also poorly coupled to mAChR-mediated increases in K+ permeability, indicating that decreased physiological sensitivity is not due to a nonspecific effect of long-term agonist exposure on general cellular function, but reflects, rather, an intrinsic property of newly synthesized mAChR. The decrease in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of mAChR on the surface nor to reduced agonist affinity of the mAChR. Cells exhibiting decreased responsiveness contain GTP-binding proteins, which function normally in the inhibition of adenylate cyclase and appear to be identical to pertussis toxin substrates from control cells using gel electrophoresis; therefore, the decreased sensitivity does not appear to be the result of an alteration in coupling proteins. These cells also contain mAChR that do not differ from those in control cells either by molecular weight or isoelectric point. Thus, the diminished sensitivity observed in cells containing newly synthesized receptors is either caused by a small change in mAChR not detected by these electrophoretic techniques or by a change in an as-yet-undefined component of mAChR transduction system in the heart.

The expression and developmental regulation of the alpha and beta subunits of the guanine nucleotide binding regulatory proteins, Gi and Go, were examined in rat atria and ventricles. Protein levels were determined by quantitative immunoblot analysis using affinity purified monospecific antibodies. Northern blot and dot blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein subunits. The concentrations of Go alpha, Gi alpha, and beta subunit protein were found to be greater in adult atrial than in adult ventricular membranes (5.2-, 1.5-, and 2.8-fold, respectively). A corresponding 3.4-fold difference in Go alpha mRNA level was also observed, as well as a 1.3-fold difference in Gi alpha-3 mRNA level. No difference was seen between the amount of beta, Gi alpha-1, Gi alpha-2 mRNA in adult atria and adult ventricles. Comparison of neonatal and adult tissues revealed a developmental decrease in ventricular Gi alpha protein and Gi alpha-2 mRNA levels (70 and 47%, respectively). Developmental decreases were also observed in the amount of mRNA encoding beta and Go alpha in ventricles (47 and 61%, respectively), and beta and Gi alpha-2 in atria (40 and 36%, respectively), while a developmental increase in atrial Gi alpha-3 mRNA levels was observed (57%). These results demonstrate differences in the expression of G protein subunits in rat atria and ventricles, as well as regulation of the levels of these subunits during cardiac development.

The regulation of the number and function of the muscarinic receptors has been investigated in cultured chick cardiac cells and in cells expressing cloned genes encoding mammalian, Drosophila, and chick muscarinic receptors. A serum-free defined medium for the culture of chick embryonic heart cells has been used to study the regulation of mAChR number and function by serum lipoproteins. Addition of rooster high density lipoprotein to the culture medium results in an attenuation of muscarinic receptor-mediated inhibition of cAMP accumulation without a change in the number of receptors or inhibitory G proteins. Clones encoding the mouse m1 receptor and a homologous receptor from Drosophila have been isolated. When expressed in Y1 adrenal cells, both receptors stimulate phosphoinositide hydrolysis but do not inhibit cAMP accumulation. Deletion of 123 out of the 156 amino acids in the third cytoplasmic loop of the mouse m1 receptor does not impair its ability to stimulate phosphoinositide hydrolysis. A genomic clone encoding a muscarinic receptor expressed in chick heart has been isolated. When expressed in Y1 cells, it causes inhibition of cAMP accumulation but does not stimulate phosphoinositide hydrolysis.