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BACKGROUND - beta-Adrenergic receptor (beta-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. beta(2)-ARs may couple to a G(i)-mediated signaling pathway that can oppose the actions of adenylyl cyclase.
METHODS AND RESULTS - In ARVMs, beta-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. beta-AR-stimulated apoptosis was abolished by the beta(1)-AR-selective antagonist CGP 20712A (P<0.05 versus beta-AR stimulation alone) but was potentiated by the beta(2)-AR-selective antagonist ICI 118,551 (P<0.05 versus beta-AR stimulation alone). The muscarinic agonist carbachol also prevented beta-AR-stimulated apoptosis (P<0.05 versus beta-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of beta-AR stimulation (P<0.05 versus beta-AR stimulation alone) and prevented the antiapoptotic action of carbachol.
CONCLUSIONS - In ARVMs, stimulation of beta(1)-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of beta(2)-ARs inhibits apoptosis via a G(i)-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.
Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.
p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. We have introduced a mutation (Ser36 --> Asn) into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro, both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induced a phenotype of rounded cells with long, beaded extensions similar to that seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, had no discernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 required GAP function. A GAP-defective mutant, p190(R1283A), had no effect on cell morphology. Moreover, the beaded extension phenotype could be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors was inhibited by wild type HA-p190, but JNK activity was enhanced by the S36N mutant. Co-expression of HA-p190 with a fragment containing only the mutated GTP binding domain partially inhibited the beaded extension phenotype, suggesting that it may sequester a factor required for p190 function. Taken together these data demonstrate that within the cell, the Rho/Rac GAP activity of p190 can be regulated by the N-terminal GTP binding domain.
Evidence suggests that cholinergic input to the hippocampus plays an important role in learning and memory and that degeneration of cholinergic terminals in the hippocampus may contribute to the memory loss associated with Alzheimer's disease. One of the more prominent effects of cholinergic agonists on hippocampal physiology is the potentiation of N-methyl-D-aspartate (NMDA)-receptor currents by muscarinic agonists. Here, we employ traditional pharmacological reagents as well as m1-toxin, an m1 antagonist with unprecedented selectivity, to demonstrate that this potentiation of NMDA-receptor currents in hippocampal CA1 pyramidal cells is mediated by the genetically defined m1 muscarinic receptor. Furthermore, we demonstrate the colocalization of the m1 muscarinic receptor and the NR1a NMDA receptor subunit at the electron microscopic level, indicating a spatial relationship that would allow for physiological interactions between these two receptors. This work demonstrates that the m1-muscarinic receptor gene product modulates excitatory synaptic transmission, and it has important implications in the study of learning and memory as well as the design of drugs to treat neurodegenerative diseases such as Alzheimer's.
We have developed a system for the co-culture of embryonic chick heart cells obtained from embryos at 3.5 days in ovo with ciliary ganglia from chick embryos at 7 days in vivo. After 3 days of co-culture, removal of the ciliary ganglia resulted in complete degeneration of axons within 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsive to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropic response to muscarinic stimulation (paired t test, P < 0.02) which persisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increase in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha 39 (39 kDa subunit) and a 2.5-fold increase in the level of alpha 41. The level of the G-protein subunit alpha s remained unchanged. Culture of embryonic chick heart cells at 3.5 days in ovo with medium conditioned by the growth of embryonic chick heart cells and ciliary ganglia had an effect on the chronotropic response to muscarinic stimulation and on alpha 39 and alpha 41 levels identical to that of co-culture. These data suggest that a soluble factor released during the co-culture of embryonic chick heart cells and ciliary ganglia is capable of inducing muscarinic responsiveness. These studies suggest that innervation of the heart may induce parasympathetic responsiveness by increasing the availability of G-proteins which couple the muscarinic receptor to a physiological response.
The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.
The causes of respiratory distress in O2 toxicity are not well understood. The purpose of this study was to better define the airway abnormalities caused by breathing 100% O2. Sheep were instrumented for measurements of dynamic compliance (Cdyn), functional residual capacity by body plethysmography (FRC), hemodynamics, and lung lymph flow. Each day Cdyn and FRC were measured before, during, and after the application of 45 min continuous positive airway pressure (CPAP) at 15 cmH2O. The amount of aerosol histamine necessary to reduce Cdyn 35% from baseline (ED35) was measured each day as was the response to aerosol metaproterenol. Cdyn decreased progressively from 0.083 +/- 0.005 (SE) 1/cmH2O at baseline to 0.032 +/- 0.004 l/cm H2O at 96 h of O2. Surprisingly, FRC did not decrease (1,397 +/- 153 ml at baseline vs. 1,523 +/- 139 ml at 96 h). The ED35 to histamine did not vary among days or from air controls. Metaproterenol produced a variable inconsistent increase in Cdyn. We also measured changes in Cdyn during changes in respiratory rate and static pressure-volume relationships in five other sheep. We found a small but significant frequency dependence of compliance and an increase in lung stiffness with O2 toxicity. We conclude that in adult sheep O2 toxicity reduces Cdyn but does not increase airway reactivity. The large reduction in Cdyn in O2 toxicity results from processes other than increased airway reactivity or reduced lung volume, and Cdyn decreases before the development of lung edema.
We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.
(2-Hydroxyethyl) methyldiethylammonium iodide (diethylcholine; DEC) was tested against trihexyphenidyl for its ability to block tremors in two animal models of Parkinsonism tremors. Both DEC (75 mg/kg) and trihexyphenidyl (10 mg/kg) antagonized physostigmine tremors in mice. Both drugs also blocked tremors in rats which received intracaudate injections of carbachol. DEC was more efficacious than trihexyphenidyl in the rat model. No dose-related inhibition of tremors was seen for trihexyphenidyl (5--20 mg/kg) but inhibition by DEC was dose-related (25--50 mg/kg). The ED50 for tremor inhibition in the rat model by DEC was 33 mg/kg. DEC was also shown to cross the blood-brain barrier in mice. The probable mechanism of action of DEC is discussed.