Search

[beta]-arrestin 1 and 2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic [beta] cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating [beta] cell function and whole-body glucose homeostasis. Initially, we inactivated the Barrl gene in [beta] cells of adult mice ([beta]-barr1-KO mice). [beta]-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, 2 widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and coimmunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in [beta] cells may prove useful for the development of efficacious antidiabetic drugs., Introduction Accumulating evidence suggests that [beta]-arrestins play key roles in regulating many important metabolic functions including [beta] cell activity (1). The 2 [beta]-arrestin isoforms ([beta]-arrestin 1 and 2; referred to [...]

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M3 and cooperative activity of RSC M[subscript 1] and M[subscript 3], and (3) memory retrieval is more impaired by inactivation of multiple M[subscript 1]-M[subscript 4] mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach.

An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic [G.sub.s]-linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic [G.sub.i] signaling. Specifically, we used a chemogenetic approach to selectively activate [G.sub.i]-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic [G.sub.i] signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased [G.sub.i] signaling stimulated glucose release in human hepatocytes. A lack of functional [G.sub.i]- type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic [G.sub.i] signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic G-coupled GPCRs may prove beneficial as antidiabetic drugs., Introduction Type 2 diabetes (T2D) has emerged as a major health problem worldwide (1). In addition to other pathophysiological features, T2D is characterized by an increase in hepatic glucose production [...]

An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein [beta]-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific [beta]-arrestin 2 deficiency did not affect hepatic insulin sensitivity or p -adrenergic signaling. Adult mice lacking [beta]-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of [beta]-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic [beta]-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes., Introduction One key pathophysiological feature of type 2 diabetes (T2D) is an increase in hepatic glucose production (HGP) (1-3). HGP is modulated by the activity of several GPCRs, including hepatic [...]

Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M,R) exhibited enhanced neurite outgrowth, confirming the role of M,R in tonic suppression of axonal plasticity. M,R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M,R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetesinduced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible., Introduction The innervation territory of intraepidermal nerve fibers (IENF) within the skin is plastic and maintained through a combination of collateral sprouting and regeneration that is regulated partly by neurotrophic [...]

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD., Introduction Due to an aging population, it is predicted that by 2040, neurodegenerative diseases, such as Alzheimer's disease (AD), will be the second most common cause of morbidity in the [...]

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the [β.sub.2] adrenergic receptor ([β.sub.2]AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the [β.sub.2]AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors., Muscarinic acetylcholine receptors (M1-M5) are GPCRs that regulate the activity of a diverse array of central and peripheral functions in the human body, including the parasympathetic actions of acetylcholine (1). [...]

Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic β cell function are predicted [...]

Thyroid hormone is a well-known regulator of metabolic and cardiovascular functions, and signaling through thyroid receptors has differential effects on cells depending on the receptor isoform that they express. In [...]

Acetylcholine, the first neurotransmitter to be identified (1), exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate (2-4). Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences (5,6). We describe here the structure of the [G.sub.q/11]-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the [G.sub.i/o]-coupled M2 receptor (7), offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en routeto the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors., The mAChR family consists of five subtypes, M1-M5, which can be subdivided into two major classes (Fig. 1a). The M1, M3 and M5 receptors show selectivity for G proteins of [...]

The activity of G protein-coupled receptors is regulated via hyper-phosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the [M.sub.3]-muscarinic receptor, a prototypical [G.sub.q/11]-coupled receptor. This mutant mouse strain was used here to investigate the role of [M.sub.3]-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to [G.sub.q/11]-signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and [beta]-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that [M.sub.3]-muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of [beta]-arrestin to the phosphorylated [M.sub.3]-muscarinic receptor. In conclusion, our findings support the unique concept that [M.sub.3]-muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/ arrestin-dependent coupling of the receptor to protein kinase D1. G-protein coupled receptor | ligand bias doi/ 10.1073/pnas.1011651107

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic [M.sub.1]-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the [M.sub.3]-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that [M.sub.3]-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the [M.sub.3]-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the [M.sub.3]-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient [M.sub.3]-muscarinic receptor was coupled normally to [G.sub.q/11]-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that [M.sub.3]-muscarinic receptor--dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased [M.sub.3]-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders. fear conditioning | G protein-coupled receptor | hippocampus | ligand bias | Alzheimer's disease doi: 10.1073/pnas.0914801107

Cholinergic bronchoconstriction is mediated by [M.sub.2] and [M.sub.3] muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-[beta]-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in [M2R.sup.-/-] and [M3R.sup.-/-] mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling. bronchus; caveolin; fluorescence resonance energy transfer; smooth muscle; muscarinic doi: 10.1152/ajplung.00261.2009.

Therapeutic strategies that augment insulin release from pancreatic [beta]-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of [beta]-cell [M.sub.3] muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through [beta]-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the [G.sub.q] family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other [beta]-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic [beta]-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with [beta]-cell-specific M3R knockout mice showed that these responses were mediated by [beta]-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic p-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes. knockout mice/muscarinic receptor/RGS proteins/G protein-coupled receptor doi/ 10.1073/pnas.1003655107

Impaired functioning of pancreatic [beta] cells is a key hallmark of type 2 diabetes. [beta] cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific [beta] cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that [beta] cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific [beta] cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in [beta] cells only. Importantly, the two designer receptors differed in their G protein-coupling properties ([G.sub.q/11] versus [G.sub.s]). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either[beta] cell [G.sub.q/11] or [G.sub.s] G proteins. Here we report the findings that conditional and selective activation of [beta] cell [G.sub.q/11] signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated [beta] cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of [beta] cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of [beta] cell function in vivo. beta cells | G protein-coupled receptors | transgenic mice | type 2 diabetes www.pnas.org/cgi/doi/10.1073/pnas.0906593106

X-linked nephrogenic diabetes insipidus (XNDI) is a severe kidney disease caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene that result in the loss of renal urine-concentrating ability. At present, no specific pharmacological therapy has been developed for XNDI, primarily due to the lack of suitable animal models. To develop what we believe to be the first viable animal model of XNDI, we generated mice in which the V2R gene could be conditionally deleted during adulthood by administration of 4-OH-tamoxifen. Radioligand-binding studies confirmed the lack of V2R-binding sites in kidneys following 4-OH-tamoxifen treatment, and further analysis indicated that upon V2R deletion, adult mice displayed all characteristic symptoms of XNDI, including polyuria, polydipsia, and resistance to the antidiuretic actions of vasopressin. Gene expression analysis suggested that activation of renal EP4 [PGE.sub.2] receptors might compensate for the lack of renal V2R activity in XNDI mice. Strikingly, both acute and chronic treatment of the mutant mice with a selective EP4 receptor agonist greatly reduced all major manifestations of XNDI, including changes in renal morphology. These physiological improvements were most likely due to a direct action on EP4 receptors expressed on collecting duct cells. These findings illustrate the usefulness of the newly generated V2R mutant mice for elucidating and testing new strategies for the potential treatment of humans with XNDI., Introduction In nephrogenic diabetes insipidus (NDI), the kidney is unable to conserve water despite normal or increased plasma levels of the antidiuretic hormone arginine vasopressin (AVP) (1-5). Most patients (> [...]

Membrane depolarization activates voltage-dependent [Ca.sup.2+] channels (VDCCs) inducing [Ca.sup.2+] release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced [Ca.sup.2+] release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local [Ca.sup.2+] release, leading to a significant increase in the activity of [Ca.sup.2+] sparks and contraction in airway SMCs. The increased [Ca.sup.2+] sparks are independent of VDCCs and the associated extracellular [Ca.sup.2+] influx. This format of local [Ca.sup.2+] release results from a direct activation of G protein-coupled, [M.sub.3] muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate ([IP.sub.3]), inducing initial [Ca.sup.2+] release through [IP.sub.3] receptors and then further [Ca.sup.2+] release via RyR2 due to a local [Ca.sup.2+]-induced [Ca.sup.2+] release process. These findings demonstrate an important mechanism for [Ca.sup.2+] signaling and attendant physiological function in SMCs. muscarinic receptors | ryanodine receptors

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the [M.sub.3] muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from [M.sub.3] receptor/ GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) [M.sub.3] receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central [M.sub.3] receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells. anterior pituitary gland | dwarfism | knockout mice

Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the [M.sub.3] muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.

Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we employed transcriptome-wide expression analysis to study gene expression in V2 vasopressin receptor (Avpr2)-deficient mice, an animal model for X-linked NDI. Gene expression changes in NDI mice indicate increased proximal tubular sodium reabsorption. Expression of several key genes including [Na.sup.+]-[K.sup.+]-ATPase and carbonic anhydrases was increased at the mRNA levels and accompanied by enhanced enzyme activities. In addition, altered expression was also observed for components of the eicosanoid and thyroid hormone pathways, including cyclooxygenases and deiodinases, in both kidney and hypothalamus. These effects are likely to contribute to the clinical NDI phenotype. Finally, our data highlight the involvement of the renin-angiotensin-aldosterone system in NDI pathophysiology and provide clues to explain the effectiveness of diuretics and indomethacin in the treatment of NDI. vasopressin receptor; diabetes insipidus; G protein-coupled receptor; signal transduction; hypernatremia

Suppressed parasympathetic nervous system (PSNS) function has been found in a variety of cardiovascular diseases, such as hypertension, heart failure, and diabetes. However, whether impaired PSNS function plays a significant role in ventricular dysfunction remains to be investigated. Cardiac regulation by the PSNS is primarily mediated by the [M.sub.2] muscarinic acetylcholine receptor ([M.sub.2]-AChR). In this study, we tested the hypothesis that lack of [M.sub.2]-AChR-mediated PSNS function may adversely impact cardiac ventricular function. Using [M.sub.2]-AChR knockout (KO) and wild-type (WT) mice, we found that the basal levels of heart rate and left ventricular function were similar in [M.sub.2]-AChR KO and WT mice. A bolus injection of isoproterenol (Iso) induced a greater increase in heart rate in [M.sub.2]-AChR KO mice than in WT mice. However, the responses of change in pressure over time (dP/dt) to Iso were similar in the two groups. After chronic infusion with Iso for 1 wk, the baseline values of left ventricular function were increased to similar extents in [M.sub.2]-AChR KO and WT mice. However, the [M.sub.2]-AChR KO mice exhibited impaired ventricular function, indicated as attenuated dP/dt and increased end-diastolic pressure, during an increase in cardiac afterload induced by a bolus injection of phenylephrine. Furthermore, chronic Iso infusion significantly increased matrix metalloproteinase (MMP) activity in the heart in [M.sub.2]-AChR KO mice. In primary culture of mixed neonatal rat cardiac fibroblast and cardiomyocytes, cotreatment with muscarinic agonist bethanechol reversed phenylephrine-induced increase in MMP-9 activation. These data suggest that [M.sub.2]-AChR may mediate an inhibitory regulation on MMP function. The overall results from this study suggest that [M.sub.2]-AChR-mediated PSNS function may provide cardiac protection. Lack of this protective mechanism will increase the susceptibility of the heart to cardiac stresses. muscarinic; cardiac; contractility; matrix metalloproteinase; isoproterenol

Thromboxane [A.sub.2] (TX[A.sub.2]) is a potent lipid mediator released by platelets and inflammatory cells and is capable of inducing vasoconstriction and bronchoconstriction. In the airways, it has been postulated that TX[A.sub.2] causes airway constriction by direct activation of thromboxane prostanoid (TP) receptors on airway smooth muscle cells. Here we demonstrate that although TX[A.sub.2] can mediate a dramatic increase in airway smooth muscle constriction and lung resistance, this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor (mAChR) antagonists. Further analyses employing pharmacological and genetic strategies demonstrate that TP-dependent changes in lung resistance and airway smooth muscle tension require expression of the [M.sub.3] mAChR subtype. These results raise the possibility that some of the beneficial actions of anticholinergic agents used in the treatment of asthma and chronic obstructive pulmonary disease result from limiting physiological changes mediated through the TP receptor. Furthermore, these findings demonstrate a unique pathway for TP regulation of homeostatic mechanisms in the airway and suggest a paradigm for the role of TX[A.sub.2] in other organ systems. nerve; prostanoid; bronchoconstriction; asthma; vagus

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the [M.sub.3] receptor subtype ([M.sub.3]R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of [M.sub.1] receptor ([M.sub.3]R) in rat chief cells. We used mice that were deficient in either the [M.sub.1] ([M.sub.3][R.sup.-/-]) or [M.sub.3] ([M.sub.3][R.sup.-/-]) receptor or that lacked both receptors ([M.sub.1/3][R.sup.-/-]) to determine the role of [M.sub.1]R and [M.sub.3]R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from [M.sub.1][R.sup.-/-] mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing [M.sub.3][R.sup.-/-] with WT glands. To explore the possibility that both [M.sub.1]R and [M.sub.3]R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from [M.sub.1/3][R.sup.-/-] double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from [M.sub.1/3][R.sup.-/-] mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine [M.sub.1]R and [M.sub.3]R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of [M.sub.1] and [M.sub.3] receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion. carbamylcholine; cholecystokinin; chief cells; gastric glands; knockout mice; in situ hybridization

To test the hypothesis that keratinocyte (KC) migration is modulated by distinct muscarinic acetylcholine (ACh) receptor subtypes, we inactivated signaling through specific receptors in in vitro and in vivo models of reepithelialization by subtype-selective antagonists, small interfering RNA, and gene knockout in mice. KC migration and wound reepithelialization were facilitated by [M.sub.4] and inhibited by [M.sub.3]. Additional studies showed that [M.sub.4] increases expression of 'migratory' integrins [[alpha].sub.5][[beta].sub.1], [[alpha].sub.v][[beta].sub.5], and [[alpha].sub.v][[beta].sub.6], whereas [M.sub.3] up-regulates 'sedentary' integrins [[alpha].sub.2][[beta].sub.1] and [[alpha].sub.3][[beta].sub.1]. Inhibition of migration by [M.sub.3] was mediated through [Ca.sup.2+]-dependent guanylyl cyclase-cyclic GMP-protein kinase G signaling pathway. The [M.sub.4] effects resulted from inhibition of the inhibitory pathway involving the adenylyl cyclasecyclic AMP-protein kinase A pathway. Both signaling pathways intersected at Rho, indicating that Rho kinase provides a common effector for [M.sub.3] and [M.sub.4] regulation of cell migration. These findings offer novel insights into the mechanisms of ACh-mediated modulation of KC migration and wound reepithelialization, and may aid the development of novel methods to promote wound healing.

Muscarinic acetylcholine receptors (mAChRs) modulate the activity of an extraordinarily large number of physiological functions. Individual members of the mAChR family ([M.sub.1]-[M.sub.5]) are expressed in a complex, overlapping fashion in most tissues and cell types. However, the identification of the precise physiological roles of individual mAChR subtypes remains a challenging task because, with the exception of a few snake toxins, mAChR ligands that can activate or inhibit specific mAChR subtypes with a high degree of selectivity are not yet available. Knowledge of the specific roles of mAChR subtypes is of considerable interest for the development of novel, clinically useful mAChR ligands. In this article, recent studies of mutant mouse strains developed, using gene targeting techniques, to be deficient in one of the three [G.sub.q]-coupled mAChR subtypes ([M.sub.1], [M.sub.3] and [M.sub.5]) are discussed. These investigations have led to many important new insights into the physiological roles of these receptor subtypes.

Author(s): Masahisa Yamada [1, 2]; Tsuyoshi Miyakawa [3, 4]; Alokesh Duttaroy [1]; Akihiro Yamanaka [5]; Toru Moriguchi [6]; Ryosuke Makita [2]; Masaharu Ogawa [2]; Chieh J. Chou [7]; Bing Xia [...]

An in situ disulfide cross-linking strategy was employed to monitor ligand-induced structural changes in a series of cycteine (Cys)-substituted mutant [M.sub.3] muscarinic acetylcholine receptors. The results provide insight into the dynamic changes that accompany [M.sub.3] receptor activation and molecular level difference between the receptor conformations induced by full and inverse muscarinic agonists.

The analysis revealed that disulfide cross-linking pattern was more restricted with the solubilized receptor proteins, both in the absence and in the presence of the muscarinic agonist, carbachol. Thus, the carbachol stimulated the formation of disulfide bonds in only two of the 10 analyzed mutant muscarinic receptors, I169C(super 3.54)/K484C(super 6.29) and I169C(super 3.54)/A4884C(super 6.33), consistent with an agonist induced rotation of the cytoplasmic end of TM VI.

Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGFαR+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease. [ABSTRACT FROM AUTHOR]

Adult mice communicate by emitting ultrasonic vocalizations (USVs) during the appetitive phases of sexual behavior. However, little is known about the genes important in controlling call production. Here, we study the induction and regulation of USVs in muscarinic and dopaminergic receptor knockout (KO) mice as well as wild-type controls during sexual behavior. Female mouse urine, but not female rat or human urine, induced USVs in male mice, whereas male urine did not induce USVs in females. Direct contact of males with females is required for eliciting high level of USVs in males. USVs (25 to120 kHz) were emitted only by males, suggesting positive state; however human-audible squeaks were produced only by females, implying negative state during male-female pairing. USVs were divided into flat and frequency-modulated calls. Male USVs often changed from continuous to broken frequency-modulated calls after initiation of mounting. In M2 KO mice, USVs were lost in about 70-80% of the mice, correlating with a loss of sexual interaction. In M5 KO mice, mean USVs were reduced by almost 80% even though sexual interaction was vigorous. In D2 KOs, the duration of USVs was extended by 20%. In M4 KOs, no significant differences were observed. Amphetamine dose-dependently induced USVs in wild-type males (most at 0.5 mg/kg i.p.), but did not elicit USVs in M5 KO or female mice. These studies suggest that M2 and M5 muscarinic receptors are needed for male USV production during male-female interactions, likely via their roles in dopamine activation. These findings are important for the understanding of the neural substrates for positive affect. [ABSTRACT FROM AUTHOR]

Thromboxane A2 (TXA2) is a potent lipid mediator released by platelets and inflammatory cells and is capable of inducing vasoconstriction and bronchoconstriction. In the airways, it has been postulated that TXA2 causes airway constriction by direct activation of thromboxane prostanoid (TP) receptors on airway smooth muscle cells. Here we demonstrate that although TXA2 can mediate a dramatic increase in airway smooth muscle constriction and lung resistance, this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor (mAChR) antagonists. Further analyses employing pharmacological and genetic strategies demonstrate that TP-dependent changes in lung resistance and airway smooth muscle tension require expression of the M3 mAChR subtype. These results raise the possibility that some of the beneficial actions of anticholinergic agents used in the treatment of asthma and chronic obstructive pulmonary disease result from limiting physiological changes mediated through the TP receptor. Furthermore, these findings demonstrate a unique pathway for TP regulation of homeostatic mechanisms in the airway and suggest a paradigm for the role of TXA2 in other organ systems. [ABSTRACT FROM AUTHOR]