Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages.

LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.7 cells) were treated with LPS in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, CaM, Gi proteins, and NF-kappaB translocation or transcription/translation. LPS effects on CaMKII phosphorylation and the expression of relevant adenylyl cyclase (AC) isoforms were measured. LPS caused a significant dose (5-10,000 ng/ml)- and time (1-8 h)-dependent increase in forskolin-stimulated AC activity that was abrogated by pretreatment with SN50 (an NF-kappaB inhibitor), actinomycin D, or cycloheximide, indicating that the effect is mediated via NF-kappaB-dependent transcription and new protein synthesis. Furthermore, LPS decreased the phosphorylation state of CaMKII, and pretreatment with a CaM antagonist attenuated the LPS-induced sensitization of AC. LPS, cAMP, or PKA activation each independently decreased macrophage proliferation. However, inhibition of NF-kappaB had no effect on LPS-induced decreased proliferation, indicating that LPS-induced decreased macrophage proliferation can proceed via PKA-independent signaling pathways. Taken together, these findings indicate that LPS induces sensitization of AC activity by augmenting the stimulatory effect of CaM and attenuating the inhibitory effect of CaMKII on isoforms of AC that are CaMK sensitive.

Actin depolymerization via the beta-adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway.

Actin is a major functional and structural cytoskeletal protein that mediates such diverse processes as motility, cytokinesis, contraction, and control of cell shape and polarity. While many extracellular signals are known to mediate actin filament polymerization, considerably less is known about signals that mediate depolymerization of the actin cytoskeleton. Human airway smooth muscle cells were briefly exposed to isoproterenol, forskolin, or the cAMP-dependent protein kinase A (PKA) agonist stimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphate (Sp-cAMPS). Actin polymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, forskolin, or Sp-cAMPS induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. The PKA inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) completely inhibited forskolin-stimulated depolymerization, whereas it only partially inhibited isoproterenol-induced depolymerization. The protein tyrosine kinase inhibitors genistein or tyrphostin A23 also partially inhibited isoproterenol-induced actin depolymerization. In contrast, the combination of Rp-cAMPS and either tyrosine kinase inhibitor had an additive effect at inhibiting isoproterenol-induced actin depolymerization. These results suggest that both PKA-dependent and -independent pathways mediate actin depolymerization in human airway smooth muscle cells.

Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells.

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric G(i) protein (the Ggamma subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.

Oxytocin and lysophosphatidic acid induce stress fiber formation in human myometrial cells via a pathway involving Rho-kinase.

The actin cytoskeleton is important for stress fiber formation and contributes to the initiation and maintenance of smooth muscle contraction. To determine if oxytocin and lysophosphatidic acid (LPA) induce stress fiber formation, cultured human myometrial cells were exposed to oxytocin (10(-5) M) or LPA (10(-6) M), and filamentous (F) and globular (G) actin pools were stained with fluorescein isothiocyanate-phalloidin and Texas red DNase I, respectively. The F- to G-actin fluorescent-staining ratio was measured by fluorescence microscopy. Oxytocin and LPA increased stress fiber formation, as indicated by an increase in the F- to G-actin fluorescent-staining ratio. The Rho-kinase inhibitor Y-27632 markedly attenuated this increase. Oxytocin-induced stress fiber formation was completely inhibited in the presence of the oxytocin antagonist compound VI. Tyrosine kinase inhibition with tyrphostin A23 partially blocked the increase induced by oxytocin but had no effect on LPA-induced stress fiber formation. Stress fiber formation was not blocked by pertussis toxin, mitogen-activated protein kinase, or protein kinase C inhibitors. Our results show that human myometrial cells respond to oxytocin and LPA with the formation of stress fibers that may be involved in the maintenance of uterine contractions. Rho-kinase appears to be a key signaling factor in this pathway.

Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment.

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either G(s)alpha or adenylyl cyclase. Detergent-solubilized G(s)alpha from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) when reconstituted into S49 cyc(-) membranes that lack endogenous G(s)alpha (carbachol pretreated: GTPgammaS, 93 +/- 13% of control; NaF/AlCl(3), 99 +/- 8.6% of control; n = 4). Exogenous G(s)alpha solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 +/- 10% of control; NaF/AlCl(3), 54 +/- 11% of control; n = 4). [(3)H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 +/- 40 and 107 +/- 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.

TNF-alpha increases transcription of Galpha(i-2) in human airway smooth muscle cells.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has an important role in the regulation of airway smooth muscle tone and reactivity. We have shown previously that TNF-alpha upregulates the expression of Galpha(i-2) protein without significantly increasing G(s)alpha protein and enhances adenylyl cyclase inhibition by carbachol in cultured human airway smooth muscle cells (Hotta K, Emala CW, and Hirshman CA. Am J Physiol Lung Cell Mol Physiol 276: L405-L411, 1999). The present study was designed to investigate the molecular mechanisms by which TNF-alpha upregulates Galpha(i-2) protein in these cells. TNF-alpha pretreatment for 48 h increased the expression of Galpha(i-2) protein without significantly altering the Galpha(i-2) protein half-life (41.0 +/- 8.2 h for control and 46.8 +/- 5.2 h for TNF-alpha-treated cells). Inhibition of new protein synthesis by cycloheximide blocked the increase in Galpha(i-2) protein induced by TNF-alpha. Furthermore, TNF-alpha treatment for 12-24 h increased the steady-state level of Galpha(i-2) mRNA without significantly altering Galpha(i-2) mRNA half-life (9.0 +/- 0.75 h for control and 8.9 +/- 1.1 h for TNF-alpha-treated cells). The transcription inhibitor actinomycin D blocked the increase in Galpha(i-2) mRNA induced by TNF-alpha. These observations indicate that the increase in Galpha(i-2) protein induced by TNF-alpha is due to an increased rate of Galpha(i-2) protein synthesis, most likely as a consequence of the transcriptional increase in the steady-state levels of its mRNA.

Cellular signaling by the potent bronchoconstrictor endothelin-1 in airway smooth muscle.

OBJECTIVE: The objective of this study was to determine whether the potent bronchoconstrictor endothelin-1 was coupled to the activation of the inositol phosphate and/or inhibition of the cyclic adenine monophosphate second messenger pathways in porcine airway smooth muscle. DESIGN: Prospective, controlled, in vitro, nonblinded study. SETTING: University biochemical and molecular biological research laboratory. SUBJECTS: Pigs of both genders. INTERVENTIONS: Airway smooth muscle was dissected from the trachea of pigs exsanguinated under anesthesia. Airway smooth muscle from six animals preloaded with 3H-myoinositol was exposed to endothelin-1, carbachol (positive control) or vehicle for 30 mins. Some tissues were pretreated with antagonists selective for the ET(A) (BQ-485) and ET(B) (BQ-788) endothelin receptor subtypes. Newly synthesized 3H-inositol phosphates were recovered by column chromatography. Airway smooth muscle from an additional 7 pigs was homogenized and incubated in the presence of 32P-alpha-adenosine triphosphate, guanosine triphosphate (GTP) and either carbachol or endothelin to measure the inhibitory influence of carbachol (positive control) or endothelin on GTP-stimulated adenylyl cyclase activity. Newly synthesized 32P-cyclic adenosine monophosphate was isolated by sequential column chromatography over Dowex and alumina. MEASUREMENTS AND MAIN RESULTS: Total inositol phosphates increased in porcine airway smooth muscle in response to either carbachol or endothelin. The endothelin receptor antagonist BQ-485 (ET(A) selective) but not BQ-788 (ET(B) selective) dose-dependently inhibited endothelin-1 induced inositol phosphate accumulation. In adenylyl cyclase assays, carbachol (positive control), but not endothelin-1, significantly inhibited GTP-stimulated adenylyl cyclase activity. CONCLUSION: Endothelin-1 couples to the activation of the inositol phosphate pathway via the ET(A) receptor subtype but does not couple to inhibition of the adenylyl cyclase pathway in porcine airway smooth muscle. The potent bronchoconstrictive effects of endothelin likely involve the acute activation of the inositol phosphate pathway in airway smooth muscle.

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat.

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.

Cholinesterase activity is similar in C3H/HeJ and A/J mice and does not account for their different bronchoconstrictor responsiveness.

Significant interstrain variation in airway responsiveness to acetylcholine (ACh) exists in inbred mouse strains. We hypothesized that part of the variation may be due to between-strain differences in cholinesterase activity. We asked if administration of neostigmine (an acetylcholinesterase inhibitor) and/or succinylcholine (an agent which competes for and inhibits butyrylcholinesterase) altered ACh responsiveness in hyporesponsive C3H/HeJ and hyperresponsive A/J mouse strains. Airway responses to ACh were measured by the airway pressure time index in the presence and absence of succinylcholine (10 mg/kg) and/or neostigmine (0.7 mg/kg). In addition, acetylcholinesterase and butyrylcholinesterase activity were directly measured. Acetylcholinesterase and butyrylcholinesterase inhibition increased airway responses to acetylcholine in both strains, but did not eliminate or decrease the differences in airway responsiveness to ACh previously seen in the two strains. Cholinesterase activities in the two strains were not significantly different. We conclude that differences in either acetylcholinesterase or butyrylcholinesterase in the A/J or C3H/HeJ mouse strains are unlikely to contribute to the differences in airway responsiveness to exogenously administered cholinergic agonists.

Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho.

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 +/- 0.3 in control cells, 6.7 +/- 0.8 with carbachol, 7.2 +/- 0.8 with LPA, and 7.4 +/- 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Galphai-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Galphai-2 and G(q)alpha were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Galphai-2 pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Galphai-2 or a Gqalpha pathway.

Gialpha but not gqalpha is linked to activation of p21(ras) in human airway smooth muscle cells.

Airway smooth muscle hypertrophy contributes to the narrowing of asthmatic airways. Activation of the mitogen-activated protein kinases is an important event in mediating cell proliferation. Because the monomeric G protein p21(ras) is an important intermediate leading to activation of mitogen-activated protein kinases, we questioned which heterotrimeric G protein-coupled receptors were linked to the activation of p21(ras) in cultured human airway smooth muscle and which of the heterotrimeric G protein subunits (alpha or betagamma) transmitted the activation signal. Carbachol and endothelin-1 increased GTP-bound p21(ras) in a pertussis toxin-sensitive manner [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): control, 30 +/- 1.7; 3 min of 1 microM carbachol, 39 +/- 1.1; 3 min of 1 microM endothelin-1, 40 +/- 1.2], whereas histamine, bradykinin, and KCl were without effect. Transfection of an inhibitor of the G protein betagamma-subunit [the carboxy terminus (Gly495-Leu689) of the beta-adrenoceptor kinase 1] failed to inhibit the carbachol-induced activation of p21(ras). These data suggest that Gi- but not Gq-coupled receptors activate p21(ras) in human airway smooth muscle cells, and this effect most likely involves the alpha-subunit.

Role of M2 muscarinic receptors in airway smooth muscle contraction.

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization by acetylcholine (ACh) and endothelin-1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca2+] in strips permeabilized with Staphylococcal alpha-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca2+]. Pretreatment with pertussis toxin for 18-20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPgammaS. We conclude that the M2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca2+] via the heterotrimeric G-protein Gi.

TNF-alpha upregulates Gialpha and Gqalpha protein expression and function in human airway smooth muscle cells.

Chronic inflammation is a characteristic feature of asthma. Multiple inflammatory mediators are released within the asthmatic lung, some of which may have detrimental effects on signal transduction pathways in airway smooth muscle. The effects of tumor necrosis factor (TNF)-alpha on the expression and function of muscarinic receptors and guanine nucleotide-binding protein (G protein) alpha-subunits were examined in human airway smooth muscle cells. Cultured human airway smooth muscle cells were incubated in serum-free culture medium for 72 h in the presence and absence of 10 ng/ml of TNF-alpha, after which the cells were lysed and subjected to electrophoresis and Galphai-2, Gqalpha, and Gsalpha protein subunits were detected by immunoblot analysis with specific antisera. TNF-alpha treatment for 72 h significantly increased the expression of Galphai-2 and Gqalpha proteins and enhanced carbachol (10(-7) M)-mediated inhibition of adenylyl cyclase activity and inositol phosphate synthesis. These data provide new evidence demonstrating that TNF-alpha not only increases expression of Galphai-2 and Gqalpha proteins but also augments the associated signal transduction pathways that would facilitate increased tone of airway smooth muscle.

Hypocapnia-induced contraction of porcine airway smooth muscle.

Hypocapnia constricts peripheral airways in vivo. This study investigated the role of airway smooth muscle in this phenomenon and the mechanism of hypocapnia-induced contraction in vitro. Isometric tension, intracellular pH (pHi) and intracellular free calcium concentration ([Ca2+]i) were measured in porcine airway smooth muscles suspended in organ baths in the presence of 5% or 0% CO2. In tracheal strips precontracted with carbachol, hypocapnic challenge (0% CO2) produced increases in tension, pHi, and [Ca2+]i. In bronchial rings or tracheal strips precontracted with carbachol, nifedipine administered between consecutive contractions attenuated responses to hypocapnia (75+/-11% above carbachol-precontracted tension before nifedipine versus 39+/-9% after nifedipine, n=7 bronchial rings, p<0.05). Neither indomethacin (5 microM), nordihydroguaiaretic acid (10 microM) nor phenidone (10 microM) significantly altered responses. These data suggest that enhanced Ca2+ influx through voltage-dependent Ca2+ channels of airway smooth muscle cells is important in airway responses to hypocapnia.

Galphai-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells.

To determine which heterotrimeric G protein couples muscarinic receptors to stress fiber formation [measured by an increase in the filamentous (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (ASM) cells, cultured human ASM cells expressing the M2 muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 microM antisense oligonucleotides designed to specifically bind to the mRNA encoding Galphai-2, Galphai-3, or Gqalpha. A randomly scrambled oligonucleotide served as a control. F- to G-actin ratios were measured with dual-fluorescence labeling after 5 min of carbachol exposure, which is known to increase the F- to G-actin ratio. Cells in parallel wells were harvested for immunoblot analysis of G protein alpha-subunit expression. Oligonucleotide antisense treatment decreased protein expression of the respective G protein alpha-subunit. Antisense depletion of the Galphai-2 protein but not of Galphai-3 or Gqalpha protein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the Galphai-2 protein couples muscarinic receptors to stress fiber formation in ASM.

Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells.

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 x 10(-4) M) had effects similar to those of oxytocin pretreatment (1 x 10(-5) M). PKC depletion with phorbol 12-myristate 13-acetate (1 x 10(-6) M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 x 10(-5) M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 microg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.

Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle.

Increased sensitivity to intracellular Ca2+ concentration ([Ca2+]) is an important mechanism for agonist-induced contraction of airway smooth muscle, but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization with acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth muscle by measuring contractions at a constant [Ca2+] in strips permeabilized with alpha-toxin or beta-escin. The peptide inhibitor G protein antagonist 2A (GP Ant-2A), which has selectivity for Gq over Gi, inhibited contractile responses to ET-1, ACh, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but the proportional inhibition of ACh responses was less than that of ET-1. Pretreatment with pertussis toxin reduced ACh contractions but had no effect on those of ET-1 or GTPgammaS. Clostridium botulinum C3 exoenzyme, which inactivates Rho family monomeric G proteins, caused similar reductions in contractile responses to ACh, ET-1, and GTPgammaS. Farnesyltransferase inhibition, which inhibits Ras G proteins, reduced responses to ET-1. We conclude that the heterotrimeric G proteins Gq and Gi both contribute to Ca2+ sensitization by ACh, whereas ET-1 responses involve Gq but not Gi. Both Gq and Gi pathways likely involve Rho family small G proteins. A Ras-mediated pathway also contributes to Ca2+ sensitization by ET-1 in airway smooth muscle.

Adenylyl cyclase messenger ribonucleic acid in myometrium: splice variant of type IV.

Regulation of uterine tone during pregnancy and parturition involves G protein-coupled receptors that modulate cellular levels of cAMP. Cyclic AMP is synthesized by a family of at least nine adenylyl cyclase enzymes that have unique tissue distributions, basal activities, and modes of regulation. Little is known regarding the expression of adenylyl cyclase isoforms in myometrium. Reverse transcription-polymerase chain reaction (PCR) was performed using RNA extracted from freshly dissected rat uteri and a cell line of homogeneous rat myometrial cells. Using PCR primers specific for adenylyl cyclases I-IX, mRNA encoding adenylyl cyclases II-IX were identified in both fresh and cultured myometrial cells. A splice variant of adenylyl cyclase type IV was also identified in RNA from both sources. These data show that multiple isoforms of adenylyl cyclases are expressed at the mRNA level in myometrial cells and suggest that the regulation of cellular levels of cAMP likely involves a complex integration of cellular second messengers acting upon multiple isoforms of adenylyl cyclases.

Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells.

To determine whether M2 muscarinic receptors are linked to the monomeric G protein Rho, we studied the effect of carbachol on actin reorganization (stress fiber formation) in cultured human airway smooth muscle cells that expressed mainly M2 muscarinic receptors by dual-fluorescence labeling of filamentous (F) and monomeric (G) actin. F-actin was labeled with FITC-labeled phalloidin, and G-actin was labeled with Texas Red-labeled DNase I. Carbachol stimulation induced stress fiber formation (increased F-actin staining) in the cells and increased the F- to G-actin ratio 3.6 +/- 0.4-fold (mean +/- SE; n = 5 experiments). Preincubation with pertussis toxin, Clostridium C3 exoenzyme, or tyrosine kinase inhibitors reduced the carbachol-induced increase in stress fiber formation and significantly decreased the F- to G-actin ratio, whereas a mitogen-activated protein kinase inhibitor, a phosphatidylinositol 3-kinase inhibitor, and a protein kinase C inhibitor were without effect. This study demonstrates that in cultured human airway smooth muscle cells, muscarinic-receptor activation induces stress fiber formation via a pathway involving a pertussis-sensitive G protein, Rho proteins, and tyrosine phosphorylation.

Neuromuscular relaxants as antagonists for M2 and M3 muscarinic receptors.

BACKGROUND: Neuromuscular relaxants such as pancuronium bind to M2 and M3 muscarinic receptors as antagonists. Blockade of muscarinic receptors in atria of the M2 subtype mediates tachycardia. In the lung, blockade of M2 receptors on parasympathetic nerves potentiates vagally induced bronchospasm, whereas blockade of M3 receptors on bronchial smooth muscle inhibits bronchospasm. The current study was designed to quantify the affinity of a series of neuromuscular relaxants for the M2 and M3 muscarinic receptors, which were individually stably transfected in Chinese hamster ovary cell lines. METHODS: Competitive radioligand binding assays determined the relative binding affinities of the neuromuscular relaxants pancuronium, succinylcholine, mivacurium, doxacurium, atracurium, rocuronium, gallamine, and pipecuronium for the muscarinic receptor in the presence of a muscarinic receptor antagonist (3H-QNB) in membranes prepared from cells individually expressing either the M2 or M3 muscarinic receptor. RESULTS: All muscle relaxants evaluated displaced 3H-QNB from muscarinic receptors. The relative order of potency for the M2 muscarinic receptor (highest to lowest) was pancuronium, gallamine, rocuronium, atracurium, pipecuronium, doxacurium, mivacurium, and succinylcholine. The relative order of potency for the M3 muscarinic receptor (highest to lowest) was pancuronium, atracurium, pipecuronium, rocuronium, mivacurium, gallamine, succinylcholine, and doxacurium. CONCLUSIONS: All neuromuscular relaxants studied had affinities for the M2 and M3 muscarinic receptor, but only pancuronium and gallamine had affinities within the range of concentrations achieved with clinical use. The high affinities of gallamine and pancuronium for the M2 muscarinic receptor are consistent with a mechanism of M2 receptor blockade in relaxant-induced tachycardia.