CHO-M1 Cells Research Articles

Hydroxycinnamic acid amides from Scopolia tangutica inhibit the activity of M1 muscarinic acetylcholine receptor in vitro

Scopolia tangutica Maxim (S. tangutica) extracts have been traditionally used as antispasmodic, sedative, and analgesic agents in Tibet and in the Qinghai province of China. Their active compositions are however poorly understood. We have recently isolated five new hydroxycinnamic acid (HCA) amides along with two known HCA amides, one cinnamic acid amide from these extracts. In this study, we evaluate their abilities to inhibit carbacol-induced activity of M1 muscarinic acetylcholine receptor along with the crude extracts. Chinese hamster ovary cells stably expressing the recombinant human M1 receptor (CHO-M1 cells) were employed to evaluate the anticholinergic potentials. Intracellular Ca2+ changes were monitored using the FLIPR system. Five HCA amides as well as the crude S. tangutica extract displayed dose-dependent inhibitory effects against M1 receptor. These findings demonstrate that HCA amides are part of the M1 receptor-inhibiting principles of S. tangutica. Since blockade of parasympathetic nerve impulse transmission through the inhibition of the M1 receptor lessens smooth muscle spasms, our findings provided a molecular explanation for the traditional use of S. tangutica against spasm.

Comparison of subcellular distribution and functions between exogenous and endogenous M1 muscarinic acetylcholine receptors

AimsRecombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors. Main methodsRecombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7). Key findingsM1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca2+ mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca2+ mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs. SignificanceExogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin.

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

Mammalian cells are extensively used for production of biopharmaceuticals. Most cells used in industry have infinite proliferative capacity, which provides a high number of cells and corresponding productivity. However, infinite cells will continue to multiply even after cell density reaches sufficient levels. This excess proliferation aggravates the culture environment and induces low productivity. Therefore, after cell density reaches sufficient levels, downregulation of proliferation would prevent such aggravation and extend the culture period and improve productivity. To realize such suitable proliferation, we aimed to establish a novel cell line whose proliferation was spontaneously downregulated after reaching a sufficient population level. Mutagenesis using high-energy beam irradiation was used. CHO-DP12 cells were irradiated with 2.5Gy X-rays and screened with hydroxyurea and 5-fluorouracil to eliminate any cells multiplying after confluence and to concentrate desired mutants. One clone was established and named CHO-M1. Cell cycle analysis indicated that CHO-M1 cells had a similar cell cycle profile in the exponential growth phase, but cells rapidly accumulated in G1 phase just before confluence and did not progress through the cell cycle. This suggested that until confluence, proliferation of CHO-M1 was similar to parental CHO, but after confluence, it was inhibited and under G1 arrest. The specific antibody production rate of CHO-M1 was kept high, even after confluence, while that of parental CHO was drastically decreased in stationary phase. These results suggest that the desired cell line was successfully established and that high-energy beam irradiation could be an efficient mutagenic technique for breeding industrial cells.

Abundance, distribution, mobility and oligomeric state of M2 muscarinic acetylcholine receptors in live cardiac muscle

M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~1μm−2, increasing at birth (to ~3μm−2) and decreasing back to ~1μm−2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening.

Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.

Intracellular Delivery of Bioactive Molecules using Light-Addressable Nanocapsules

This paper describes a method by which molecules that are impermeable to cells are encapsulated in dye-sensitized lipid nanocapsules for delivery into cells via endocytosis. Once inside the cells, the molecules are released from the lipid nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645 nm). We demonstrate this method with the intracellular release of the second messenger IP(3) in CHO-M1 cells and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average number of IP(3) released is decreased below 50 molecules per nanocapsule. We also demonstrate the delivery of a 23 kDa O(6)-alkylguanine-DNA alkyltransferase (AGT) fusion protein into Ba/F3 cells to inhibit a key player BCR-ABL in the apoptotic pathway. We show that an average of ∼8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells.

The snake venom peptide Bj‐PRO‐7a is a M1 muscarinic acetylcholine receptor agonist

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (<EDGPIPP) specifically activates [Ca(2+)](i) transients in CHO-M1 cells expressing heterologous rat M1 muscarinic subtype. The activation curve established by microfluorimetry in CHO-M1 cells using increasing concentrations of Bj-PRO-7a reached the maximum response in the presence of 3 μM Bj-PRO-7a (EC(50) = 0.25 ± 0.07 μM). The variation observed by calcium imaging in these cells ranged from 52 to 1218 nM (EC(50) = 0.31 ± 0.12 μM). [Ca(2+)](i) responses in CHO-M1 cells were largely inhibited by pirenzepine, a specific M1 antagonist. Neural-differentiated P19 cells expressing endogenous M1 receptors were also responsive to Bj-PRO-7a application, whereas no such response was observed in undifferentiated P19 cells not expressing muscarinic receptors. As further support for its specific action on M1 receptors, the peptide did not activate M3 subtypes in transfected CHO cells. Our findings provide a novel M1 muscarinic receptor agonist that could be used for basic research and even for pharmacological applications.

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

The idea that GPCRs (G-protein-coupled receptors) may exist as homo- or hetero-oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype). We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.

Role of CREB in the regulatory action of sarsasapogenin on muscarinic M 1 receptor density during cell aging

This work studied the role of cyclic AMP responsive element binding protein (CREB) in the up-regulation of M 1 muscarinic acetylcholine receptor (M 1 receptor) density by sarsasapogenin (ZMS) in CHO cells transfected with M 1 receptor gene (CHOm1 cells). During cell aging, sarsasapogenin elevated M 1 receptor density as well as CREB and phosphor-CREB (pCREB) levels. CREB peaked earliest, followed by pCREB and M 1 receptor density peaked last. When CREB synthesis was blocked by antisense oligonucleotides, the elevation effect of sarsasapogenin on M 1 receptor density was abolished. These results suggest that sarsasapogenin up-regulates M 1 receptor density in aged cells by promoting CREB production and phosphorylation. Furthermore, the results support the hypothesis that pCREB regulates M 1 receptor gene expression through heterodimer formation.

Effects of Chlorpyrifos Oxon on M2 Muscarinic Receptor Internalization in Different Cell Types

The muscarinic M2 receptor is a member of the G protein-coupled receptor (GPCR) superfamily. Agonist activation of GPCR leads to their phosphorylation, desensitization, internalization, and subsequent endocytic recycling or lysosomal degradation. Agonist-induced phosphorylation of M2 receptors is mediated by G-protein receptor kinase 2 (GRK2). The active metabolite of the organophosphorus insecticide chlorpyrifos, i.e., chlorpyrifos oxon (CPO), inhibited agonist-induced phosphorylation of human recombinant M2 receptors by GRK2 in vitro in a concentration-dependent manner. In both intact HEL 299 cells (human embryonic lung fibroblasts expressing M2 receptors) and CHO-M2 cells (stably expressing M2 receptors), the muscarinic agonist carbachol (100 μM) led to receptor internalization as determined by reduced specific binding to the membrane-impermeable radioligand [3H]-N-methylscopolamine (NMS). CPO alone (100 μM) exerted no significant effect on NMS binding in either HEL 299 or CHO-M2 cells. In HEL 299 cells, CPO did not influence carbachol-induced internalization, whereas in CHO-M2 cells CPO blocked internalization. In primary striatal neurons, M2 receptors appeared widely and diffusely distributed. Exposure to either carbachol or CPO led to apparent receptor internalization with an increased percent of cells exhibiting punctate domains of immunostaining, while combined exposure to both carbachol and CPO led to a significantly higher percent of cells exhibiting this appearance. The data suggest that CPO may differentially influence agonist-stimulated M2 receptor internalization in a cell-dependent manner.

Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17

Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFα-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.

An investigation of whether agonist‐selective receptor conformations occur with respect to M2 and M4 muscarinic acetylcholine receptor signalling via Gi/o and Gs proteins

1. A range of muscarinic acetylcholine (mACh) receptor agonists (methacholine (MCh), oxotremorine-M (OXO-M), oxotremorine (OXO), arecoline (AREC), bethanechol (BETH), pilocarpine (PILO)) have been investigated with respect to their binding to, and activation of, M(2) and M(4) mACh receptors, recombinantly expressed in Chinese hamster ovary cells, to explore the possibility that these agonists may differentially affect mACh receptor-G(i/o) and -G(s) coupling. 2. M(2)/M(4) mACh receptor coupling to the adenylyl cyclase/cyclic AMP signalling pathway has been explored in intact cells. G(i/o)-mediated negative coupling to adenylyl cyclase was explored functionally by assessing the ability of the mACh receptor agonists to inhibit forskolin-stimulated enzymic activity. Following pertussis toxin treatment (100 ng ml(-1), 18-20 h) to inactivate G(i/o) proteins, each agonist caused a G(s)-mediated enhancement of forskolin-stimulated adenylyl cyclase activity. 3. At both M(2) and M(4) mACh receptors, all agonists tested were more potent in mediating G(i/o)- versus G(s)-coupled responses. This difference (determined as the pIC(50) (G(i/o) coupling) minus pEC(50) (G(s) coupling) value) was greatest for AREC (65-75-fold) and least for BETH and PILO (</=10-fold). 4. Using apparent binding affinities (pK(B)), and potency (EC(50)/IC(50)) and responsiveness (E(max)/I(max)) estimates, relative efficacy (e(rel)) values for each agonist with respect to M(2) and M(4) mACh receptor coupling to G(i/o)- and G(s)-mediated signalling were also calculated. While the e(rel) values obtained for MCh and OXO-M in CHO-m2 cells were similar, OXO-M behaved as a 'super-agonist' at the M(4) mACh receptor giving greater e(rel) values for both G(i/o) and G(s) coupling relative to MCh. 5. The experimental data indicate that while interesting differences between agonists with respect to M(2)/M(4) mACh receptor activation and receptor-G(i/o) and -G(s) coupling can be discerned, no clear examples of agonist trafficking of signal have emerged.

Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches.

To determine whether autoantibodies directed against muscarinic M3 receptors are present in the serum of patients with primary Sjogren's syndrome (pSS), and if so whether these autoantibodies inhibit secretion by intact salivary acinar cells. IgG was purified by affinity chromatography using protein G from sera collected from 15 patients with pSS. Antibody detection was by Western blotting, whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The antisecretory activity of the IgG faction was determined using fura-2 microfluorimetry to measure changes in intracellular Ca(2+) activity ([Ca(2+)](i)) in human and mouse salivary gland acinar cells and in Chinese hamster ovary (CHO) cells transfected with human M3 receptors (CHO-M3). We found no specific M3 receptor recognition by the IgG fraction obtained from pSS patients using either Western blotting or ELISA or immunoblot techniques in which epitope conformation were preserved. Chronic exposure to pSS IgG had no effect on agonist-evoked Ca(2+) signals measured in human or mouse submandibular acinar cells or in CHO-M3 cells. However, acute application of IgG from Sjogren's syndrome patients produced a rapidly reversible reduction in the agonist-stimulated elevation in [Ca(2+)](i). These data represent the first demonstration of salivary acinar cell inhibition by pSS IgG; however, this inhibition was found to be reversible. Our data also show that pSS IgG binding to M3R cannot be visualized by conventional immunological approaches.

Rac1 Activation Drives a Nuclear ARM Race

Lanning et al. investigated the involvement of RhoA and Rac1 in nuclear transport of the armadillo (ARM) protein SmgGDS and discovered a new role for these two small guanosine triphosphatases (GTPases), as well as a novel mechanism for nuclear transport of ARM proteins. Many ARM family proteins shuttle between the nucleus and cytoplasm, regulating events in both of these compartments. Some ARM proteins contain nuclear localization signal (NLS) sequences or associate with proteins containing NLS sequences; others enter the nucleus through mechanisms that are unclear. SmgGDS is an ARM protein that promotes guanine nucleotide exchange in GTPases with a C-terminal polybasic region (PBR). These include RhoA and Rac1, which are involved in regulating the actin cytoskeleton. Noting the resemblance of the PBR to an NLS, Lanning et al. demonstrated that both fluorescently labeled Rac1 PBR and fluorescently labeled Rac1 accumulated in the nucleus when expressed in CHO-m3 cells. Mutational analysis indicated that Rac1 activation, as well as an intact PBR, promoted nuclear accumulation of Rac1 and of hemagglutinin (HA)-tagged SmgGDS. Rac1 with a mutated PBR promoted nuclear accumulation of SmgGDS less efficiently than did wild-type Rac1 and a dominant negative (DN) Rac1 mutant was ineffective. DN Rac1 containing a mutated PBR diminished accumulation of SmgGDS in the nucleus. Fluorescently labeled RhoA PBR or RhoA showed little nuclear accumulation and inhibited nuclear accumulation of SmgGDS. The authors developed a model whereby SmgGDS associates with and activates Rac1; Rac1 activation and the PBR then promote movement of the complex into the nucleus. RhoA competitively inhibits the association with Rac1, thereby diminishing nuclear accumulation. C. C. Lanning, R. Ruiz-Velasco, C. L. Williams, Novel mechanism of the co-regulation of nuclear transport of SmgGDS and Rac1. J. Biol. Chem . 278 , 12495-12506 (2003). [Abstract] [Full Text]

Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells.

1. We have examined possible mechanisms of cross-talk between the G(q/11)-linked M(3) muscarinic acetylcholine (mACh) receptor and the G(i/o)-linked M(2) mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P(3)) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, expressing comparable levels of M(2) or M(3) mACh receptors. 2. Based on comparisons between cell-lines and pertussis toxin (PTx) pretreatment to eliminate receptor-G(i/o) coupling, evidence was obtained for (i) an M(2) mACh receptor-mediated contribution to the predominantly M(3) mACh receptor-mediated Ins(1,4,5)P(3) response and (ii) a facilitation of the inhibitory effect of M(2) mACh receptor on forskolin-stimulated cyclic AMP accumulation by M(3) mACh receptor coactivation at low agonist concentrations (MCh 10(-9)-10(-6) M). 3. The most profound cross-talk effects were observed with respect to ERK activation. Thus, while MCh stimulated ERK activation in both CHO-m2 and CHO-m3 cells (pEC(50) values: 5.64+/-0.09 and 5.57+/-0.16, respectively), the concentration-effect relation was approx 50-fold left-shifted in CHO-m2m3 cells (pEC(50): 7.17+/-0.07). In addition, the ERK response was greater and more sustained in CHO-m2m3 cells. In contrast, only minor differences were seen in the time-courses and concentration-dependencies of JNK activation in CHO-m3 and CHO-m2m3 cells. 4. Costimulation of endogenous P2Y(2) purinoceptors also caused an approx 10-fold left-shift in the MCh-stimulated ERK response in CHO-m2 cells, suggesting that the G(q/11)/G(i/o) interaction to affect ERK activation is not specific to muscarinic receptors. 5. PTx pretreatment of cells had unexpected effects on ERK activation by MCh in both CHO-m2m3 and CHO-m3 cells. Thus, in CHO-m3 cells PTx pretreatment caused a marked left-shift in the MCh concentration-effect curve, while in PTx-treated CHO-m2m3 cells the maximal responsiveness was decreased, but the potency of MCh was only slightly affected. 6. The data presented here strongly suggest that cross-talk between M(2) and M(3) mACh receptors occurs at the level of both second messenger and ERK regulation. Further, these data provide novel insights into the involvement of G(i/o) proteins in both positive and negative modulation of ERK responses evoked by G protein-coupled receptors.

Interaction of intravenous anesthetics with recombinant human M1-M3 muscarinic receptors expressed in chinese hamster ovary cells.

Previous reports suggest that the effects of propofol, ketamine, and thiopental on airway tone may be because of modulation of parasympathetic activity. We examined if these anesthetics interact with recombinant human M1-M3 muscarinic receptors expressed in Chinese hamster ovary cells (CHO-M1, M2, and M3) using the displacement of 0.4 nM of l-[N-methyl-(3)H]scopolamine methyl chloride([(3)H]NMS). In addition, functional studies were performed by fluorometrically monitoring methacholine (1 mM) stimulated intracellular Ca(2+) ([Ca(2+)](i)) responses. Ketamine concentration dependently displaced [(3)H]NMS binding to CHO-M1, M2, and M3 cells with affinity, pK(i) (mean K(i)) values of 4.34 +/- 0.14 (45 micro M), 3.53 +/- 0.10 (294 micro M), and 3.61 +/- 0.02 (246 micro M), respectively. The effects at M1 were in the clinical range. Ketamine did not affect either basal or methacholine stimulated increase in [Ca(2+)](i) in CHO-M1 cells. Thiopental significantly displaced [(3)H]NMS binding to M3 (pKi [mean Ki] = 4.12 +/- 0.06 [75 micro M]) but not M1 or M2 receptors. Thiopental (10(-5)-10(-3) M) concentration dependently inhibited methacholine stimulated increase in [Ca(2+)](i) in CHO-M3 cells. Propofol and barbituric acid did not interact with any muscarinic receptor subtype. We suggest that at the level of [Ca(2+)](I), thiopental may possess M3 antagonist activity, whereas there are no functional consequences of the interaction of ketamine with the M1 receptor. In this study using recombinant human M1-M3 muscarinic receptors, we show that for agonist-stimulated increases in intracellular Ca(2+) thiopental acts as a M3 antagonist.

Unique in Vivo Associations with SmgGDS and RhoGDI and Different Guanine Nucleotide Exchange Activities Exhibited by RhoA, Dominant Negative RhoAAsn-19, and Activated RhoAVal-14

We compared the in vivo characteristics of hemagglutinin (HA)-tagged RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14) stably expressed in Chinese hamster ovary (CHO) cells. Proteins co-precipitating with these HA-tagged GTPases were identified by peptide sequencing or by Western blotting. Dominant negative RhoA(Asn-19) co-precipitates with the guanine nucleotide exchange factor (GEF) SmgGDS but does not detectably interact with other expressed GEFs, such as Ost or Dbl. SmgGDS co-precipitates minimally with wild-type RhoA and does not detectably associate with RhoA(Val-14). The guanine nucleotide dissociation inhibitor RhoGDI co-precipitates with RhoA, and to a lesser extent with RhoA(Val-14), but does not detectably co-precipitate with RhoA(Asn-19). Wild-type RhoA is predominantly in the [(32)P]GDP-bound form, RhoA(Val-14) is predominantly in the [(32)P]GTP-bound form, and negligible levels of [(32)P]GDP or [(32)P]GTP are bound to RhoA(Asn-19) in (32)P-labeled cells. Immunofluorescence analyses indicate that HA-RhoA(Asn-19) is excluded from the nucleus and cell junctions. Microinjection of SmgGDS cDNA into CHO cells stably expressing HA-RhoA causes HA-RhoA to be excluded from the nucleus and cell junctions, similar to the distribution of RhoA(Asn-19). Our findings indicate that the expression of RhoA(Asn-19) may specifically inhibit signaling pathways that rely upon the SmgGDS-dependent activation of RhoA.

M3 Muscarinic Acetylcholine Receptors Regulate Cytoplasmic Myosin by a Process Involving RhoA and Requiring Conventional Protein Kinase C Isoforms

Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (alpha), novel (delta), and atypical (iota) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 microM), calphostin C (IC50 = 2.4 microM), or chelerythrine (IC50 = 8.0 microM). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation.

Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors

Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs, or stress-activated protein kinases) are activated by diverse extracellular signals and mediate a variety of cellular responses, including mitogenesis, differentiation, hypertrophy, inflammatory reactions and apoptosis. We have examined the involvement of Ca2+ and protein kinase C (PKC) in ERK and JNK activation by the human G-protein-coupled m2 and m3 muscarinic acetylcholine receptors (mAChR) expressed in Chinese hamster ovary (CHO) cells. We show that the Ca2+-mobilizing m3 AChR is efficiently coupled to JNK and ERK activation, whereas the m2 AChR activates ERK but not JNK. Activation of JNK in CHO-m3 cells by the agonist methacholine (MCh) was delayed in onset and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The EC50 values for MCh-induced ERK activation in both cell types were essentially identical and similar to that for JNK activation in CHO-m3 cells, suggesting little amplification of the response. Agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-m3 cells was insensitive to pertussis toxin (PTX), consistent with a Gq/phosphoinositide-specific phospholipase C-β mediated pathway, whereas a significant component of ERK and JNK activation in CHO-m3 cells was PTX-sensitive, indicating Gi/o involvement. Using manipulations that prevent receptor-mediated extracellular Ca2+ influx and intracellular Ca2+-store release, we also show that ERK activation by m2 and m3 receptors is Ca2+-independent. In contrast, a significant component (> 50%) of JNK activation mediated by the m3 AChR was dependent on Ca2+, mainly derived from extracellular influx. PKC inhibition and down-regulation studies suggested that JNK activation was negatively regulated by PKC. Conversely, ERK activation by both m2 and m3 AChRs required PKC, suggesting a novel mechanism for PKC activation by PTX-sensitive m2 AChRs. In summary, mAChRs activate JNK and ERK via divergent mechanisms involving either Ca2+ or PKC respectively.

Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors

Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs, or stress-activated protein kinases) are activated by diverse extracellular signals and mediate a variety of cellular responses, including mitogenesis, differentiation, hypertrophy, inflammatory reactions and apoptosis. We have examined the involvement of Ca2+ and protein kinase C (PKC) in ERK and JNK activation by the human G-protein-coupled m2 and m3 muscarinic acetylcholine receptors (mAChR) expressed in Chinese hamster ovary (CHO) cells. We show that the Ca2+-mobilizing m3 AChR is efficiently coupled to JNK and ERK activation, whereas the m2 AChR activates ERK but not JNK. Activation of JNK in CHO-m3 cells by the agonist methacholine (MCh) was delayed in onset and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The EC50 values for MCh-induced ERK activation in both cell types were essentially identical and similar to that for JNK activation in CHO-m3 cells, suggesting little amplification of the response. Agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-m3 cells was insensitive to pertussis toxin (PTX), consistent with a Gq/phosphoinositide-specific phospholipase C-beta mediated pathway, whereas a significant component of ERK and JNK activation in CHO-m3 cells was PTX-sensitive, indicating Gi/o involvement. Using manipulations that prevent receptor-mediated extracellular Ca2+ influx and intracellular Ca2+-store release, we also show that ERK activation by m2 and m3 receptors is Ca2+-independent. In contrast, a significant component (>50%) of JNK activation mediated by the m3 AChR was dependent on Ca2+, mainly derived from extracellular influx. PKC inhibition and down-regulation studies suggested that JNK activation was negatively regulated by PKC. Conversely, ERK activation by both m2 and m3 AChRs required PKC, suggesting a novel mechanism for PKC activation by PTX-sensitive m2 AChRs. In summary, mAChRs activate JNK and ERK via divergent mechanisms involving either Ca2+ or PKC respectively.