We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

• MeSH
• Adenylyl Cyclases metabolism   MeSH
• Receptors, Adrenergic, beta-3 metabolism   MeSH
• Adaptation, Physiological * genetics   MeSH
• Stress, Physiological * genetics   MeSH
• Catecholamines biosynthesis   MeSH
• Mice MeSH
• Cold Temperature * MeSH
• Receptor, Muscarinic M2 deficiency   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Heart physiopathology   MeSH
• Heart Ventricles enzymology   pathology   physiopathology   MeSH
• Nitric Oxide Synthase metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylyl Cyclases MeSH
• Receptors, Adrenergic, beta-3 MeSH
• Catecholamines MeSH
• Receptor, Muscarinic M2 MeSH
• Nitric Oxide Synthase MeSH

Mammal heart tissue has long been assumed to be the exclusive domain of the M(2) subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M(2) subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M(1) muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M(1)-M(4) receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M(2) subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M(2), with the majority of these belonging to the M(1) subtype. We were also able to detect a marginal fraction (6 +/- 2%) of receptors that, based on other findings, belong mainly to the M(5) muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M(1)-M(4) receptors and can not therefore be used as a method for M(5) muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M(1) muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M(5) muscarinic receptors but not to that of the M(3) receptors.

• MeSH
• Gene Expression MeSH
• Type C Phospholipases metabolism   MeSH
• Binding, Competitive MeSH
• Rats MeSH
• Rats, Wistar MeSH
• Receptor, Muscarinic M1 drug effects   metabolism   MeSH
• Receptor, Muscarinic M2 drug effects   metabolism   MeSH
• Receptor, Muscarinic M3 drug effects   metabolism   MeSH
• Receptor, Muscarinic M5 drug effects   metabolism   MeSH
• Receptors, Muscarinic drug effects   metabolism   MeSH
• Heart Ventricles drug effects   metabolism   MeSH
• Heart Atria drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Type C Phospholipases MeSH
• Receptor, Muscarinic M1 MeSH
• Receptor, Muscarinic M2 MeSH
• Receptor, Muscarinic M3 MeSH
• Receptor, Muscarinic M5 MeSH
• Receptors, Muscarinic MeSH

The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also alpha(1)-adrenoceptors and beta-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of alpha(1A), alpha(1B), alpha(1D), beta(1), and beta(2)-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, alpha(1)-adrenoceptors to 23%, and beta-adrenoceptors to about 50% of control. Changes were subtype specific, as alpha(1A), alpha(1B), and beta(2)-adrenoceptors, but not alpha(1D)-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.

• MeSH
• Acetylcholine metabolism   MeSH
• Acetylcholinesterase genetics   MeSH
• Receptors, Adrenergic biosynthesis   MeSH
• Cyclic AMP biosynthesis   MeSH
• Down-Regulation MeSH
• Phosphatidylinositols metabolism   MeSH
• Type C Phospholipases metabolism   MeSH
• Immunoassay MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Lung metabolism   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Radioligand Assay MeSH
• Receptors, Muscarinic biosynthesis   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Acetylcholine MeSH
• Acetylcholinesterase MeSH
• Receptors, Adrenergic MeSH
• Cyclic AMP MeSH
• Phosphatidylinositols MeSH
• Type C Phospholipases MeSH
• GTP-Binding Proteins MeSH
• Receptors, Muscarinic MeSH