The role of nitric oxide in mediating nonadrenergic, noncholinergic relaxation in rat pulmonary artery.

Experiments were undertaken to investigate the existence of inhibitory nonadrenergic, noncholinergic (i-NANC) nerve activity by using in vitro functional and immunohistochemical techniques in rat main pulmonary arterial rings. Vessels precontracted with phenylephrine (3 microM) relaxed in response to electrical field stimulation (EFS) (50 V, 0.2 ms, 0.1-10 Hz for 5 s) in the presence of atropine (1 microM) and guanethidine (1 microM). Tetrodotoxin (0.3 microM) abolished this response, indicating that it is neuronal in origin. l-NAME (30 microM), methylene blue (10 microM), and removal of endothelium significantly reduced the EFS-induced relaxations. The inhibitory action of l-NAME was completely reversed by l-arginine (1 mM) but not by d-arginine (1 mM). Moreover l-arginine alone potentiated the magnitude of the relaxations elicited by EFS. On the other hand, immunohistochemical work clearly demonstrated the existence of neuronal nitric oxide synthase in the pulmonary artery vessel wall. All these results are consistent with the suggestion that nitric oxide is the likely mediator of this vasodilatation. However, the incomplete blockade of the responses by l-NAME gives evidence of an additional inhibitory NANC neurotransmitter(s) mediating the residual relaxation, which requires further experiments to clarify its nature.     

Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function

Airway smooth muscle (ASM) plays an important role in asthma pathophysiology through its contractile and proliferative functions. The cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are second messengers capable of mediating the effects of a variety of drugs and hormones. There is a large body of evidence to support the hypothesis that cAMP is a mediator of the ASM's relaxant effects of drugs, such as beta2-adrenoceptor agonists, in human airways. Although most attention has been paid to this second messenger and the signal transduction pathways it activates, recent evidence suggests that cGMP is also an important second messenger in ASM with important relaxant and antiproliferative effects. Here, we review the regulation and function of cGMP in ASM and discuss the implications for asthma pathophysiology and therapeutics. Recent studies suggest that activators of soluble and particulate guanylyl cyclases, such as nitric oxide donors and natriuretic peptides, have both relaxant and antiproliferative effects that are mediated through cGMP-dependent and cGMP-independent pathways. Abnormalities in these pathways may contribute to asthma pathophysiology, and therapeutic manipulation may complement the effects of beta2-adrenoceptor agonists.     

Nitric oxide may be the final mediator of nonadrenergic, noncholinergic inhibitory junction potentials in the gut.

This study tested the hypothesis that the final mediator of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (ijps) and associated relaxation responses was nitric oxide (NO) or a related substance and not vasoactive intestinal polypeptide (VIP). We used opossum esophagus body circular muscle and canine intestine circular muscle. In both these tissues, ijps had reversal potentials near the potassium equilibrium potential, (EK); in esophagus the ijps were apamin insensitive, but in the intestine they were partially apamin sensitive. N omega-Nitro-L-arginine methyl ester (NAME) (10(-5) to 5 x 10(-4) M) abolished ijps in both tissues, an effect overcome by 10(-3) M L-arginine but not D-arginine. NAME increased input resistance of esophagus tissues in the double sucrose gap but caused no significant depolarization in the sucrose gap or in studies with microelectrodes. Contractions and basal tension were increased in both tissues by NAME. The apamin sensitive and insensitive ijp components in canine muscle were both abolished by NAME, but the time course of this abolition was different for the two components. Methylene blue (10-50 microM) with variable rapidity and extent inhibited ijps in both tissues, but L-arginine could not overcome this effect. Methylene blue, like NAME, did not depolarize detectably but enhanced the contractile activity. VIP (10(-6) M) had very small effects in both tissues, little or no hyperpolarization and increased input resistance in esophagus, these effects were not changed by NAME, and VIP did not affect ijps. We conclude that NO may be the final mediator of NANC-initiated inhibitory junction potentials in gastrointestinal circular smooth muscle.     

Isotonic relaxation of control and sensitized airway smooth muscle

Smooth muscle relaxation has most often been studied in isometric mode. However, this only tells us about the stiffness properties of the bronchial wall and thus only about wall capacitative properties. It tells us little about airflow. To study the latter, which of course is the meaningful parameter in regulation of ventilation and in asthma, we studied isotonic shortening of bronchial smooth muscle (BSM) strips. Failure of BSM to relax could be another important factor in maintaining high airway resistance. To analyze relaxation curves, we developed an index of isotonic relaxation, t1/2(P, lCE), which is the half-time for relaxation that is independent of muscle load (P) and of initial contractile element length (lCE). This index was measured in curves of relaxation initiated at 2 s (normally cycling crossbridges) and at 10 s (latch-bridges). At 10 s no difference was seen for adjusted t1/2(P, lCE) between curves obtained from control and sensitized BSM, (8.38 +/- 0.92 s vs. 7.78 +/- 0.93 s, respectively). At 2 s the half-time was almost doubled in the sensitized BSM (6.98 +/- 0.01 s (control) vs. 12.74 +/- 2.5 s (sensitized)). Thus, changes in isotonic relaxation are only seen during early contraction. Using zero load clamps, we monitored the time course of velocity during relaxation and noted that it varied according to 3 phases. The first phase (phase i) immediately followed cessation of electrical field stimulation (EFS) at 10 s and showed almost the same velocity as during the latter 1/3 of shortening; the second phase (phase ii) was linear in shape and is associated with zero load velocity, we speculate it could stem from elastic recoil of the cells' internal resistor; and the third phase (phase iii) was convex downwards. The zero load velocities in phase iii showed a surprising spontaneous increase suggesting reactivation of the muscle. Measurements of intracellular calcium (Fura-2 study) and of phosphorylation of the 20 kDa myosin light chain showed simultaneous increments, indicating phase iii represented an active process. Studies are under way to determine what changes occur in these 3 phases in a sensitized muscle. And of course, in the context of this conference, just what role the plastic properties of the muscle play in relaxation requires serious consideration.     

Mechanisms of airway smooth muscle relaxation during maturation

Greater airway responsiveness in healthy juveniles is considered a factor in the higher asthma prevalence at a young age compared with adults. We have developed a guinea pig maturational model that utilizes tracheal strips from 1-week-, 3-week-, and 3-month-old guinea pigs to study the role of airway smooth muscle (ASM) in juvenile airway hyperresponsiveness. Because a reduced ability of ASM to spontaneously relax may contribute to airway hyperresponsiveness by maintaining bronchospasm and thus high airway resistance, we have employed this model to study ASM spontaneous relaxation during electrical field stimulation (EFS). Since relaxation during EFS had been neither described nor quantified during maturation, we developed new indices that allowed an appropriate comparison of the relaxing response from strips of different age animals. Using these indices we found that, whereas strips from adult animals relax to a level of tension similar to that found in the absence of stimulation, this ability to spontaneously relax is essentially absent in trachealis from infant animals. These results confirmed that maturation of ASM relaxation may play a role in juvenile airway hyperresponsiveness and that our maturational model is suitable to study the mechanisms regulating spontaneous relaxation in physiological conditions. We investigated the role of prostanoids in ASM relaxation and showed that cyclooxygenase inhibition increases relaxation in infant ASM to levels similar to adults. These results suggest that prostanoids regulate the ability of ASM to spontaneously relax, i.e., they reduce relaxation. We have produced preliminary data suggesting a maturational change in the level of prostanoids. Moreover, the possible action of acetylcholinesterase on maturation of ASM relaxation is discussed here on the basis of a preliminary study. We suggest that impairment of ASM relaxation likely contributes to increased airway responsiveness.     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation.

Interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) and increased the cyclic GMP (cGMP) concentration in the cells. The dose dependencies of the two effects were similar (IC50 = 4 U/ml for the anti-proliferation and EC50 = 3 U/ml for cGMP formation) and the effect of IFN-gamma was enhanced by tumor necrosis factor-alpha treatment. Furthermore, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, inhibited both activities induced by IFN-gamma. These findings show that the anti-proliferation and cGMP formation are closely related and that IFN-gamma inhibits the proliferation of rat VSMC by generation of NO through the induction of an NO synthase.     

Mechanisms mediating the antiproliferative effects of nitric oxide in cultured human airway smooth muscle cells

We have characterised the mechanisms involved in the antiproliferative effect of NO in human airway smooth muscle cells (HASMC). S-Nitroso-N-acetyl penicillamine, a nitric oxide donor, inhibited proliferation in both G(1) and S phases of the cell cycle. Additionally, experiments with 8-bromo-cGMP, haemoglobin, a NO scavenger and zaprinast, a cGMP-specific phosphodiesterase inhibitor, showed that both effects were NO-mediated. The G(1) phase inhibition was cGMP-dependent whereas the S phase inhibition was due to a cGMP-independent inhibition of ribonucleotide reductase. These results demonstrate that NO inhibits HASMC proliferation by cGMP-dependent and -independent mechanisms acting at distinct points in the cell cycle.     

Nitric oxide as a nonadrenergic inhibitory transmitter in smooth muscle cells of the guinea pig gastro-intestinal tract: Mechanisms of action

The role of nitric oxide (NO) as a possible transmitter for nonadrenergic inhibitory transmission was studied on isolated muscle strips of the guinea pig gastro-intestinal tract (GIT) using sucrose-gap technique. In addition, the voltage clamp and intracellular dialysis techniques were employed to study the effects of sodium nitroprusside (NP) on isolated smooth muscle (SM) cells of thetaenia coli. N-nitro-L-arginine methyl ester (L-NAME), a blocker of NO synthesis from L-arginine (0.1 mM), was shown to selectively suppress the apamin-resistant component of nonadrenergic inhibitory junctional (synaptic) potentials (IJP) in the guinea pig GIT SM cells. At the same time, L-NAME did not affect the vasoactive intestinal polypeptide (VIP)- and NP-evoked hyperpolarization in SM cells of the colon. The NP-induced hyperpolarization (0.1 mM) was accompained by a decrease in the SM cell membrane resistance. Application of NP to isolated SM cells activated a small outward current and increased the frequency of spontaneous transient calcium-dependent outward currents. NP increased the Ca-dependent potassium current evoked in SM cells by step depolarization, but did not affect the potassium currents of delayed rectification. Our results suggest that NO is involved in generation of nonadrenergic IJP in SM cells of the guinea pig GIT. The action of NP on SM cells is complex and results in hyperpolarization and relaxation (partially through the activation of Ca-dependent potassium channels in SM cell membrane).     

cGMP-independent mechanism of airway smooth muscle relaxation induced by S-nitrosoglutathione

This study tested the hypothesis that the NO donorS-nitrosoglutathione (GSNO) relaxescanine tracheal smooth muscle (CTSM) in part by a cGMP-independentprocess that involves reversible oxidation of intracellular thiols.GSNO caused a concentration-dependent relaxation in ACh-contractedstrips (EC₅₀ ~1.2 µM)accompanied by a concentration-dependent increase in cytosolic cGMPconcentration ([cGMP]_i). Thesoluble guanylate cyclase inhibitor methylene blue prevented theincrease in [cGMP]_iinduced by 1 and 10 µM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of[cGMP]_i to baseline,GSNO-induced relaxation persisted during continuous ACh stimulation.Dithiothreitol caused a rapid recovery of isometric force to valuessimilar to those obtained with ACh alone in these strips. We concludethat GSNO relaxes CTSM contracted by ACh in part by oxidation ofintracellular protein thiols.

     

Facilitation of isoproterenol induced airway smooth muscle relaxation by nifedipine

The facilitating effect of nifedipine on isoproterenol induced airway smooth muscle relaxation was studied in guinea pig tracheas. For isometric force measurement, 4 mm tracheal cylinders were suspended in incubation chambers in oxygenated physiologic medium. After 90 minutes of equilibration under 2 grams resting tension, at a temperature of 37 degrees C and pH of 7.4, concentration response curves for isoproterenol were performed with and without the addition of a 1 X 10(-5)M nifedipine dose. The experiments were then repeated using tissues precontracted with histamine. Our data show that in the nifedipine pretreated tissues, the EC50 of isoproterenol is shifted to the left (p less than 0.05) probably due to further reduction in cytosolic calcium by nifedipine. Our findings suggest that nifedipine might have a role in the treatment of asthma and obstructive airway disease.     

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.     

GABAA receptors are expressed and facilitate relaxation in airway smooth muscle

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)) channels and metabotropic (GABA(B)) receptors. GABA(A) channels are ubiquitously expressed in neuronal tissues, and in mature neurons modulate an inward chloride current resulting in neuronal inhibition due to membrane hyperpolarization. In airway smooth muscle (ASM) cells, membrane hyperpolarization favors smooth muscle relaxation. Although GABA(A) channels and GABA(B) receptors have been functionally identified on peripheral nerves in the lung, GABA(A) channels have never been identified on ASM itself. We detected the mRNA encoding of the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, delta-, gamma(1-3)-, pi-, and theta-subunits in total RNA isolated from native human and guinea pig ASM and from cultured human ASM cells. Selected immunoblots identified the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, and gamma(2)-subunit proteins in native human and guinea pig ASM and cultured human ASM cells. The GABA(A) beta(3)-subunit protein was immunohistochemically localized to ASM in guinea pig tracheal rings. While muscimol, a specific GABA(A) channel agonist, did not affect the magnitude or the time to peak contractile effect of substance P, it directly concentration dependently relaxed a tachykinin-induced contraction in guinea pig tracheal rings, which was inhibited by the GABA(A)-selective antagonist gabazine. Muscimol also relaxed a contraction induced by an alternative contractile agonist histamine. These results demonstrate that functional GABA(A) channels are expressed on ASM and suggest a novel therapeutic target for the relaxation of ASM in diseases such as asthma and chronic obstructive lung disease.     

Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle.

Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.     

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.     

cAMP, myosin dephosphorylation, and isometric relaxation of airway smooth muscle

The temporal relationships among increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels, myosin dephosphorylation, and relaxation were investigated to clarify the mechanisms of airway muscle relaxation. Canine tracheal muscles isometrically contracted (82% of maximum force) with 10(-6) M methacholine were relaxed by adding either 4 x 10(-7) M atropine or 4 x 10(-5) M forskolin. Atropine had no effect on cAMP levels; myosin phosphorylation and force, however, decayed at the same rates and these two parameters returned to their basal pre-methacholine levels within 5 min. Forskolin treatment results in about a 10-fold increase in cAMP levels; myosin phosphorylation and force decayed simultaneously to their respective steady-state levels by 10 min but neither parameter returned to its pre-methacholine level. The addition of forskolin to muscles maximally contracted with 10(-4) M methacholine leads to about a 30-fold increase in cAMP levels. However, there are minimal decreases in myosin phosphorylation and force in these muscles. Thus myosin dephosphorylation appears to be essential for airway muscle relaxation, whereas an increase in cAMP in the absence of myosin dephosphorylation is insufficient to cause relaxation. Moreover, myosin dephosphorylation appears to be a common step in the cAMP-independent and cAMP-dependent mechanisms for airway muscle relaxation.