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.

Activation of transfected M1 or M3 muscarinic acetylcholine receptors induces cell-cell adhesion of Chinese hamster ovary cells expressing endogenous cadherins.

Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell-cell adhesion is induced within 15 min of activating transfected M1 or M3 mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2 mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell-cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+ abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1 or M3 mAChR stimulation may involve cadherin activation.

Ethanol disrupts carbamylcholine-stimulated release of arachidonic acid from Chinese hamster ovary cells expressing different subtypes of human muscarinic receptor.

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [3H]arachidonic acid ([3H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [3H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [3H]AA release by up to 12-fold with an ECo of approximately 0.4 microM; maximal responses were obtained with 10 microM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [3H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [3H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [3H]AA release insofar as maximal [3H]AA release was depressed in the presence of ethanol with no apparent change in the EC50 for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [3H]AA release induced by carbamylcholine without affecting basal [3H]AA release or altering the EC50 for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.

Influence of acute and chronic ethanol treatment on muscarinic responses and receptor expression in Chinese hamster ovary cells.

The influence of ethanol on the muscarinic receptor-mediated release of inositol phosphate from Chinese hamster ovary (CHO) cells stably transfected with one of the five subtypes of muscarinic acetylcholine receptor was determined. In CHO cells expressing M3 muscarinic receptors (CHO-M3), carbamylcholine increased muscarinic receptor-induced release of inositol phosphate by 150-350% following a 15-min incubation with an EC50 of approximately 30 microM. Maximal responses were obtained with 1 mM carbamylcholine, while responses to 10 mM carbamylcholine were somewhat less than maximal. Preincubation with atropine for 10 min inhibited the response with an IC50 of approximately 30 nM. CHO cells transfected with M1, M3, and M5 receptors displayed a similar pattern of activity; CHO cells transfected with M2 and M4, as well as untransfected cells, were unresponsive to carbamylcholine. Ethanol acutely inhibited the response of CHO-M3 cells to carbamylcholine by 15% at 18 mM and by 47% at 180 mM (the highest concentration examined). CHO-M3 cells were incubated with 50 mM ethanol for 48 hr. This treatment did not affect the number of cells or their protein content (113 pg/cell). The expression of M3 muscarinic receptors (determined using [3H]N-methylscopolamine) increased from 1.34 +/- 0.23 to 1.75 +/- 0.16 pmol/mg protein (P < 0.05). In contrast, carbamylcholine-stimulated release of inositol phosphate was depressed by 40-70% in four experiments. Concentration-response analyses indicated a non-competitive inhibitory mechanism. This dissociation of muscarinic receptor expression and muscarinic signaling suggests a compensatory increase in receptor expression in response to chronic inhibition of muscarinic signaling by ethanol.

Inhibition of M3 muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpiperazin e (KN-62).

The role of Ca2+/calmodulin-dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+ concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3 mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne). Carbamylcholine (100 microM) elicited an initial transient peak in [Ca2+]i due to mobilization of Ca2+ from internal stores, followed by a sustained elevation in [Ca2+]i that depended on the influx of extracellular Ca2+ and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 microM KN-62. KN-62 inhibited mAChR-induced Ca2+ influx and mobilization from internal stores by about 25-30%, producing a half-maximal effect at approximately 1 microM. In contrast, KN-62 (25 microM) almost completely abolished carbamylcholine-stimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+ influx induced by depolarization of the cells with 25 mM K+ (IC50 = 3 microM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+ and the stimulation of Ca2+ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+ occurs through Ca2+ channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.

Poly(dA).poly(dT) forms very stable nucleosomes at higher temperatures.

The synthetic polymer poly(dA).poly(dT) was long thought to be refractory to nucleosome formation. Several years ago our laboratory demonstrated that the polymer could be mixed with authentic nucleosomes in a low-salt exchange procedure to form a nucleoprotein complex that behaved in a manner identical with that of nucleosomes. Competitive exchange assays at 37 degrees C showed that the homopolymer reconstituted about as well as heterogenous-sequence DNA. However, studies by other laboratories have shown that the conformation of poly(dA).poly(dT) depends on temperature; the polymer converts from its well-known, atypical structure, found at ambient temperature, to a conformation more closely resembling a canonical B form as temperature is increased. We have measured the ability of the homopurine.homopyrimidine to form nucleosomes as a function of temperature. It is seen that poly(dA).poly(dT) forms nucleosomes more strongly as the temperature of the exchange mixture is increased, so that poly(dA).(dT) outcompetes heterogeneous-sequence DNA for histones at elevated temperatures.

Structure of nucleosomal DNA at high salt concentration as probed by hydroxyl radical.

The structure of a 146 base-pair nucleosomal DNA has been probed using hydroxyl radical cleavage in buffers containing NaCl concentrations ranging from 80 mM to 800 mM. The highest salt concentrations used here are close to those required to dissociate core histone H2A and H2B from nucleosomal DNA. Nonetheless, the cleavage pattern of the DNA is unchanged over the tenfold salt concentration range, retaining the approximately 10.0 base-pairs per turn helical periodicity in the flanking regions and approximately 10.7 base-pairs per turn periodicity in the central dyad region that is characteristic of nucleosomal DNA. The rotational frame of the DNA is similarly unaffected by salt. These results support the contention that the differential free energy of bending of DNA around the nucleosome is independent of salt concentration.

Poly[d(A.T)] and other synthetic polydeoxynucleotides containing oligoadenosine tracts form nucleosomes easily.

Synthetic double-stranded polydeoxynucleotides of the general form poly[d(AnT).d(ATn)], with n ranging from 3 to 11, have been synthesized. The conformation of the polymers was investigated by circular dichroism spectroscopy and the polymers were examined for their ability to form nucleosomes. Although spectra show that a circular dichroism band characteristic of poly[d(A.T)] appears in the polymer family for n greater than 7, we demonstrate that even polynucleotides with the longest tracts of contiguous adenosine bases (n = 11) are able to form nucleosomes when reconstituted using a histone exchange procedure. Thus resistance to nucleosome formation does not coincide with the appearance of features similar to that of poly[d(A.T)] over the bulk of the nucleosomal DNA. Furthermore, we show that an approximately 150 base-pair poly[d(A.T)] itself, long thought to be refractory to nucleosome formation, can assemble into such a protein-DNA complex when reconstituted by a low-salt exchange procedure. Competitive assays show that the homopolymer reconstitutes about as well as heterogeneous sequences DNA. Our work, therefore, suggests that highly adenosine-rich sequences in vivo apparently have a function that operates at a level other than that of nucleosome structure.