Cyclic nucleotide-dependent protein kinases in airway smooth muscle

Because of the potential importance of cyclic nucleotide-dependent protein kinases in the regulation of airway smooth muscle tone, we have examined some of the characteristics of these enzymes in the soluble fraction of canine trachealis homogenates. In the absence of added cAMP, the heat-stable cAMP-dependent protein kinase inhibitor (PKI) abolished only a half of the 32P incorporation into mixed histones. The remaining activity appeared to be contributed by a cyclic nucleotide-independent enzyme. Phosphotransferase activity was enhanced 5-fold by 5 microM cAMP but only 70% of the cAMP-stimulated activity could be inhibited by PKI. The sensitivity of the cyclic nucleotide-dependent, PKI-resistant enzyme to cAMP, cGMP, and Mg2+ indicated that it was cGMP-dependent protein kinase. Because of the large amount of cyclic nucleotide-independent activity, and the ability of cAMP to activate cGMP-dependent protein kinase, the traditional "-cAMP/+cAMP" ratio did not provide an accurate assessment of the in vivo activation state of cAMP-dependent protein kinase. However, a modified assay was developed which allowed the precise measurement of cAMP-dependent, cGMP-dependent, and cyclic nucleotide-independent protein kinase activities. Using this new method, the cAMP-dependent protein kinase activity ratio of 0.239 in untreated trachealis strips was increased to 0.355 and 0.386 by prior exposure of the intact tissue to the smooth muscle relaxants isoproterenol and prostaglandin E2, respectively. The results of this study are consistent with the proposed role of cAMP-dependent protein kinase in the regulation of smooth muscle contractile function.     

Modulation of Ca2+ sensitivity by cyclic nucleotides in smooth muscle from protein kinase G-deficient mice

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.     

Response to acetylcholine and myosin content of isolated canine airways

Contractility of tracheal smooth muscle strips and spiral strips of fourth to fifth generation bronchi was studied in organ baths. The relationship among contractility, airway smooth muscle myosin, and smooth muscle thickness was also examined. The trachea was divided into three segments, each consisting of 12-14 rings. Smooth muscle strips from each of the three regions (top, middle, and bottom of the trachea) and from fourth to fifth generation bronchi were studied. Acetylcholine (ACh) sensitivity (-log EC50) was 8.1, 7.1, 7.9, and 6.1 for the top, middle, and bottom of the trachea and the bronchi, respectively. At P = 0.01, the EC50 ACh value of the top of the trachea differed from the EC50 value of the bronchi. Maximal tension (Tmax) generated in bronchi (3.2 g) was lower (P less than 0.01) than in the top (10.4 g), middle (7.1 g), and bottom of the trachea (5.1 g). Differences between trachea and bronchi disappeared when Tmax was corrected for smooth muscle myosin content. Thickness of smooth muscle in bronchi was less (P less than 0.01) than in the three regions of trachea. Tmax was significantly correlated with airway smooth muscle thickness (r = 0.56; P less than 0.05). These results suggest that in mongrel dogs sensitivity to ACh shows a gradient from the top of the trachea to the bronchi and that Tmax is greater in the trachea than in the bronchi and is significantly correlated with thickness of smooth muscle.     

Methacholine causes reflex bronchoconstriction

To determine whether methacholine causes vagally mediated reflexconstriction of airway smooth muscle, we administered methacholine tosheep either via the bronchial artery or as an aerosol via tracheostomyinto the lower airways. We then measured the contraction of anisolated, in situ segment of trachealis smooth muscle and determinedthe effect of vagotomy on the trachealis response. Administeringmethacholine to the subcarinal airways via the bronchial artery(0.5-10.0 µg/ml) caused dose-dependent bronchoconstriction andcontraction of the tracheal segment. At the highest methacholine concentration delivered, trachealis smooth muscle tension increased anaverage of 186% over baseline. Aerosolized methacholine (5-7 breaths of 100 mg/ml) increased trachealis tension by 58% and airwaysresistance by 183%. As the bronchial circulation in the sheep does notsupply the trachea, we postulated that the trachealis contraction wascaused by a reflex response to methacholine in the lower airways.Bilateral vagotomy essentially eliminated the trachealis response andthe airways resistance change after lower airways challenge (either viathe bronchial artery or via aerosol) with methacholine. We concludethat 1) methacholine causes asubstantial reflex contraction of airway smooth muscle and2) the assumption may not be validthat a response to methacholine in humans or experimental animalsrepresents solely the direct effect on smooth muscle.

     

Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

Mechanical properties of human bronchial smooth muscle in vitro.

The in vitro mechanical properties of smooth muscle strips from 10 human main stem bronchi obtained immediately after pneumonectomy were evaluated. Maximal active isometric and isotonic responses were obtained at varying lengths by use of electrical field stimulation (EFS). At the length (Lmax) producing maximal force (Pmax), resting tension was very high (60.0 +/- 8.8% Pmax). Maximal fractional muscle shortening was 25.0 +/- 9.0% at a length of 75% Lmax, whereas less shortening occurred at Lmax (12.2 +/- 2.7%). The addition of increasing elastic loads produced an exponential decrease in the shortening and velocity of shortening but increased tension generation of muscle strips stimulated by EFS. Morphometric analysis revealed that muscle accounted for 8.7 +/- 1.5% of the total cross-sectional tissue area. Evaluation of two human tracheal smooth muscle preparations revealed mechanics similar to the bronchial preparations. Passive tension at Lmax was 10-fold greater and maximal active shortening was threefold less than that previously demonstrated for porcine trachealis by us of the same apparatus. We attribute the limited shortening of human bronchial and tracheal smooth muscle to the larger load presumably provided by a connective tissue parallel elastic component within the evaluated tissues, which must be overcome for shortening to occur. We suggest that a decrease in airway wall elastance could increase smooth muscle shortening, leading to excessive responses to contractile agonists, as seen in airway hyperresponsiveness.     

Cyclic AMP efflux is regulated by occupancy of the adenosine receptor in pig aortic smooth muscle cells

Cultured pig aortic smooth muscle cells respond to extracellular adenosine by activating adenylate cyclase and by initiating the efflux of cAMP. In the presence of extracellular adenosine, efflux is first order with respect to intracellular cAMP concentration up to at least 125 pmol/10(6) cells. The apparent first-order rate constant for the efflux of cAMP increases in a dose-dependent manner in response to extracellular adenosine or 5-N-ethylcarboxamide adenosine. The EC50 for adenosine for promoting cAMP efflux is 12 microM. For cells stimulated with 5-N-ethylcarboxamide adenosine, the EC50 is 5 microM. When extracellular adenosine is removed, efflux stops abruptly. Cellular cAMP content falls but is still in a range that supports cAMP efflux when agonist is present. Efflux is not affected by H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP-dependent protein kinase. These data suggest that in pig aortic smooth muscle cells, the efficiency of cAMP efflux is regulated by A2 receptor occupancy.     

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside

Methacholine (3 microM) and sodium nitroprusside (300 microM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 microM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0 degree C) and with an abbreviated incubation time (2.5 min). When assayed at 30 degrees C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 microM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0 degree C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.     

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside

Methacholine (3 μM) and sodium nitroprusside (300 μM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 μM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0°C) and with an abbreviated incubation time (2.5 min). When assayed at 30°C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 μM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0°C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.     

The effect of methacholine and histamine on cyclic AMP-dependent protein kinase activity in the guinea-pig isolated trachea.

The effects of methacholine and histamine were examined on cyclic AMP-dependent protein kinase (A-kinase) activity in guinea-pig isolated trachea, using kemptide as a substrate for phosphorylation during the determination of the enzyme activity. Methacholine (EC90, 10 microM) induced a rapid reduction in the basal A-kinase activity ratio, which was maximal after 30 s. This initial reduction coincided with the early phase of isometric tension development, and returned to control levels 4 min after the addition of methacholine. Pretreatment with atropine inhibited the methacholine response. In contrast, histamine (EC90, 30 microM) was without effect upon A-kinase activity ratio. The results establish the sensitivity of the A-kinase assay using kemptide and demonstrate that not all contractile agonists have the capacity to inhibit basal activity of A-kinase in airway smooth muscle.     

Long-term hypoxia changes myometrial responsiveness and oxytocin receptors in the pregnant ewe: differential effects on longitudinal versus circular smooth muscle

Previous studies from our laboratory demonstrated that long-term hypoxia (LTH) altered in vitro contractile responses to oxytocin in full-thickness myometrial strips from pregnant sheep. The present study was designed to determine, first, if the reduced contractile response to oxytocin following LTH is the result of combined effects on longitudinal and circular smooth muscle or if the effect is specific to a single muscle layer and, second, if the reduced contractile response to oxytocin following LTH is caused by changes in oxytocin-receptor protein. Pregnant ewes were maintained at high altitude (3820 m) from Day 30 to Days 137-142 of gestation, when the ewes were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Longitudinal and circular layers were separated, length-tension curves generated to determine optimal resting tension, and all strips exposed to increasing half-log doses of oxytocin ranging from 10-12 to 10-6.5 M. The expression of oxytocin-receptor protein was measured using Western blot analysis. We found that LTH did not affect KCl-induced contraction of either smooth muscle layer, whereas the sensitivity of both myometrial layers to oxytocin was altered. A decreased maximum contractile response of the circular layer to oxytocin was also observed. Additionally, LTH decreased expression of oxytocin-receptor protein in the circular layer and increased levels in the longitudinal layer. Results from the present study indicate that LTH alters contractile responses and oxytocin-receptor protein expression in a layer-specific manner in the pregnant sheep myometrium.     

A comparison of preterm and adult airway smooth muscle mechanics

To determine whether airway smooth muscle undergoes a maturational change regarding force generation, length-tension relationships were determined in isolated trachealis strips from adult and preterm sheep. At the length of maximum force generation, passive active and total tensions of the adult muscle were 2.5 times greater than preterm values (P less than 0.001). KCl stimulation yielded a greater peak tension in the adult strips than in the preterm strips (P less than 0.01). Preterm strips required higher concentrations of KCl to initiate contractions and higher concentrations to reach peak tension. Acetylcholine- (ACh) induced contraction resulted in greater force development at each dose in the adult strips compared with preterm strips (P less than 0.001). The dose of ACh required to reach a half-maximal response was significantly less for the adult strips than for the preterm strips (P less than 0.005). These data demonstrate that both force generation and receptor sensitivity increase with age. This inability of immature smooth muscle to generate as much force as adult smooth muscle may help explain why very preterm neonates requiring intermittent positive-pressure ventilation are at risk for developing structural airway problems.     

Calcium uptake into guinea-pig trachealis: The effect of epithelium removal

Removal of the epithelium from preparations of guinea-pig airways increases the responsiveness of the smooth muscle of normal and ovalbumin-sensitized animals to a number of contractile agents. To determine if epithelium removal results in an increase in Ca²⁺ entry into the smooth muscle, the effect of removing the epithelium on Ca²⁺ uptake into the trachealis smooth muscle was studied using a modified La³⁺-technique. KCl increased Ca²⁺ uptake in the presence and absence of the epithelium in control and sensitized animals. Methacholine did not promote Ca²⁺ uptake, whether or not the epithelium was present, in either control or sensitized animals. Ovalbumin did not stimulate Ca²⁺ uptake into the trachealis of sensitized animals. These results indicate that the increase in responsiveness of airway smooth muscle seen on epithelium removal is not a consequence of a facilitation of Ca²⁺ entry into the muscle. The increased responsiveness to methacholine in control animals, and to ovalbumin in preparations in tension studies in epithelium-free tissues from sensitized animals, cannot be explained by an increased availability of extracellular Ca²⁺ into the muscle, but, rather may reflect some other effect of the epithelium-derived modulatory factor.     

Leukotriene generation and small airway smooth muscle responsiveness in human lung

The hypothesis was tested that endogenous leukotriene (LT) production in the lung causes desensitisation of airway smooth muscle to LT. The synthesis of LTB4, C4, D4 and E4 by human lung tissue, obtained at thoracotomies, after stimulation with Ca-ionophore was assessed by HPLC. Functional studies of small airway smooth muscle from the same tissue specimens were carried out using LTC4 and methacholine as the contracting agents. Generation of LTB4, C4, D4 and E4 was 453 +/- 82, 84 +/- 15, 71 +/- 27 and 40 +/- 16 pmol/g fresh tissue respectively (mean +/- S.E.M., n = 10). All airway smooth muscle preparations responded to LTC4 in a concentration dependent way with a -log EC20 of 8.56 +/- 0.13, a -log EC50 of 7.95 +/- 0.08 and a Tmax of 82 +/- 11 mg force/mg tissue weight, corresponding to 79 +/- 4% of the maximal response to methacholine (mean +/- S.E.M.; 27 preparations from 10 patients). No correlations were found between any of the functional parameters (-logEC20, -logEC50, Tmax to LTC4 and methacholine) and the amounts of LT's generated by the lung tissue. Furthermore airway smooth muscle contractility was not significantly reduced after repeated exposure of bronchiolar strips to LTC4 in vitro. These findings suggest that the responsiveness of human peripheral airway smooth muscle to LT is not related to the capacity of the lung tissue to synthetize LT.     

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.     

Phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase in intact smooth muscle

A monoclonal antibody was used to quantitate changes in the extent of phosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase in intact bovine tracheal smooth muscle. The autophosphorylated and nonphosphorylated forms of the regulatory subunit (RII) were separated in sodium dodecyl sulfate-polyacrylamide gels and identified by immunoblot analysis. Addition of cAMP to tissue extracts resulted in rapid dephosphorylation of RII (t 1/2 = 20s at 4 degrees C) while addition of MgATP caused complete conversion to the phosphorylated form. Under basal conditions, 56% of RII in intact muscle was phosphorylated when the tissue was homogenized under conditions which fully inhibit protein kinase and phosphatase activities. Incubation with isoproterenol caused a dose-dependent decrease in the phosphorylation state of RII (EC50 = 5 X 10(-8) M). Incubation with high concentrations of isoproterenol, 1-methyl-3-isobutylxanthine, or forskolin caused maximal decreases in the phosphorylated form to 12-18% of the total RII. The effect of isoproterenol was rapid (t 1/2 = 15 s at 37 degrees C), reversible, and could be blocked with the antagonist propranolol. Contraction of the smooth muscle with K+ or low (less than 1 microM) concentrations of carbachol had no effect on the phosphorylation level. A decrease in the basal phosphorylation level to 41% was observed with 10 microM carbachol which was additive with the dephosphorylation produced by isoproterenol. The time course of isoproterenol-induced dephosphorylation of RII paralleled that of muscle relaxation, consistent with a role of cAMP-dependent protein kinase activation in relaxation of smooth muscle.     

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.     

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.     

Inhibition of cAMP-dependent protein kinase by adenosine cyclic 3'-, 5'-phosphorodithioate, a second cAMP antagonist

A single sulfur substitution for either the axial or the equatorial exocyclic oxygen of adenosine cyclic 3', 5'-phosphate (cAMP) results in diastereometric phosphorothioate analogs of cAMP with agonist versus antagonist properties towards activation of cAMP-dependent protein kinase. Sulfur substitutions for both of the exocyclic oxygens of cAMP results in a dithioate analog of cAMP, adenosine cyclic 3', 5'-phosphorodithioate (cAMPS2), which has antagonist properties. cAMPS2 displaced [3H]cAMP from the binding sites on bovine heart Type II cAMP-dependent protein kinase as demonstrated by equilibrium dialysis experiments with an apparent Kd of 6.3 microM. The addition of 10, 30, or 100 microM cAMPS2 when measuring cAMP-induced activation of pure porcine heart Type II cAMP-dependent protein kinase resulted in a concentration-dependent increase in the amount of cAMP required to produce half-maximal activation (EC50). A plot of the EC50 values as a function of the cAMPS2 concentration resulted in a straight line from which a KI value of 4 microM was derived. cAMPS2 had no significant effect on the degree of cooperativity (n) of cAMP activation of the holoenzyme. These data suggest that the most important structural requirement for the dissociation of the holoenzyme is an equatorial exocyclic oxygen.     

Vasodilation by the calcium-mobilizing messenger cyclic ADP-ribose

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.