Essential role of neuronal M3 mAChRs are for normal somatic growth Several studies suggest that central muscarinic cholinergic pathways play a role in stimulating GH release in experimental animals and humans. The identification of the molecular pathways and the specific mAChR subtypes involved in mediating this activity should be of considerable potential therapeutic interest. To examine the possible role of central M3 receptors in modulating GH release, we used Cre/loxP technology to generate a new set of mutant mice, referred to as Br-M3-KO mice, that selectively lacked the M3 receptor subtype in the brain (neurons and glial cells;Gautam et al. Proc. Natl. Acad Sci. U.S.A. 106, 6398-6403, 2009.). We demonstrated that Br-M3-KO mice displayed a dramatic hypoplasia of the anterior pituitary gland, associated with greatly reduced pituitary GH and prolactin levels. Moreover, serum GH and IGF-1 levels, the major endocrine regulators of postnatal growth in mammals, were significantly decreased in these mutant mice, leading to greatly reduced longitudinal growth. Since M3 receptors are expressed in the hypothalamus at relatively high density, we speculated that hypothalamic M3 receptor activity might be required for the proper function of GHRH neurons, resulting in the observed hypoplasia of the anterior pituitary and reduced longitudinal growth of Br-M3-KO mice. In agreement with this concept, we demonstrated that treatment of Br-M3-KO mice with CJC-1295, a synthetic GHRH analog, restored normal pituitary size and serum GH and IGF-1 levels, and normal longitudinal growth. This observation suggests that the primary defect underlying the dwarf phenotype of these mutant mice does not reside in the pituitary gland itself but most likely involves impaired function of hypothalamic GHRH neurons. Consistent with this notion, we found that hypothalamic GHRH levels were greatly reduced in Br-M3-KO mice. Moreover, in situ mRNA hybridization studies showed that M3 receptors were expressed by hypothalamic GHRH neurons, the primary site of GHRH synthesis and storage. Taken together, these findings suggest that M3 mAChR activity is critical for the proper function of GHRH neurons. In summary, these studies revealed an unexpected and critical role of central M3 receptors in the proliferation of the anterior pituitary and the stimulation of longitudinal growth. Central M3 receptors may represent a potential pharmacological target to enhance or inhibit GH release in the treatment of human growth disorders. Central roles of M2 and M4 mAChRs Previously published data suggest that central M2 and M4 receptors key roles in many fundamental functions of the brain, including learning and memory, locomotor activity, and mood control. In order to learn more about the localization of the M2 and M4 receptors involved in these important central processes, we used Cre/loxP technology to generate a series of conditional mAChR mutant mice which lack M2 or M4 receptors only in specific regions of the brain. We initially generated floxed M2 or M4 receptor mutant mice in which the receptor coding sequences were flanked by loxP sites (floxed M2 or M4 receptor mutant mice). The floxed M2 and M4 receptor mice were then crossed with transgenic mice that express Cre recombinase only in certain regions of the brain (forebrain, distinct regions of the hippocampus, D1 receptor-containing cells, etc.). The resulting mice are currently being studied by various biochemical, pharmacological, and behavioral techniques. The analysis of these newly generated mutant mice should lead to novel insights into the physiological and pathophysiological roles of central M2 and M4 receptors. The following results were obtained in collaborative studies: 1. Studies with M3 receptor KO mice demonstrated that M3 receptor activity induces local calcium release in smooth muscle cells, initially through IP3 receptors and subsequently via ryanodine receptors due to local release processes. These findings shed light onto the mechanisms by which M3 receptor signaling affects calcium signaling in smooth muscle cells. Liu QH, et al. Proc. Natl. Acad Sci. U.S.A. 106, 11418-11423, 2009. 2. Smooth muscle M3 receptors are known to play a critical role in mediating bronchoconstriction. IgE/antigen-mediated increases in airway resistance depend on the release of serotonin by mast cells. We found that serotonin had little or no effect on airway resistance in M3 receptor KO mice, indicating that mast cell-derived serotonin requires the existence of muscarinic pathways to cause changes in airway patency during anaphylaxis. Cyphert JM, et al. J. Immunol. 182, 7430-7439, 2009. 3. Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. It is well known that muscarinic agonists increase particle transport speed and ciliary beat frequency. Functional studies with different mAChR KO strains demonstrated that M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. These findings are of considerable clinical interest. Klein MK, et al. Eur. Respir. J. 33, 1113-1121, 2009. 4. Studies with different mAChR KO strains demonstrated that activation of atrial M3 receptors leads to positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M2 receptor activation on atrial contractility. Kitazawa T, et al. J. Pharmacol. Exp. Ther. 330, 487-493, 2009. 5. Functional studies demonstrated that cholinergic dilation of ophthalmic arteries was almost completely abolished in M3 receptor KO mice, providing the first piece of direct experimental evidence that M3 receptors are critically involved in regulating the diameter of ophthalmic arteries. Gericke A, et al. Invest. Ophthalmol. Vis. Sci. Apr 30, 2009 Epub ahead of print. 6. Previous studies showed that male (but not female) M5 receptor KO mice suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss. We demonstrated that these deficits could be reversed or prevented by treatment of male M5 receptor KO mice with 17beta-estradiol (E2). Our findings indicate that M5 receptor KO mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. Kitamura N, et al. PLoS ONE 4, e5159, 2009. 7. Peripheral mouse tissues were immunostained for specific mAChR subtypes by using a large number of commercially available antibodies. Somewhat surprisingly, all antibodies, with only two exceptions, yielded identical labeling patterns in tissues from wild-type and mAChR KO mice. These results strongly suggest that tissues from mAChR KO mice need be used to verify the specificity of antibodies employed to visualize specific mAChR subtypes. Jositsch G, et al. Naunyn Schmiedebergs Arch. Pharmacol. 379, 389-395, 2009.
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