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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

S-nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea pig

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.     

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca²⁺ signaling, and Ca²⁺ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca²⁺]_i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca²⁺ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca²⁺ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca²⁺ oscillations. NOC-5 inhibited the increase of [Ca²⁺]_i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP₃) in airway SMCs. The effect of NO on the Ca²⁺ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca²⁺ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca²⁺-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca²⁺ via the IP₃ receptor to decrease the frequency of agonist-induced Ca²⁺ oscillations.     

Release of nitric oxide from human vascular smooth muscle cells

It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NO-release from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10(-9) moles nitric oxide per 10(8) cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10(-5) M) to 3% of the control levels (p less than 0.02, n = 4), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.     

Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation.

In this study, we examined effects of chemical stimulation of the ventrolateral region of the midbrain periaqueductal gray (vl PAG) on airway smooth muscle tone. We observed that in anesthetized, paralyzed, and artificially ventilated ferrets, vl PAG stimulation elicited airway smooth muscle relaxation. To clarify the mechanisms underlying this observation, we examined the GABA-GABA(A) receptor signaling pathway by 1) examining the expression of GABA(A) receptors on airway-related vagal preganglionic neurons (AVPNs) located in the rostral nucleus ambiguus region (rNA), by use of receptor immunochemistry and confocal microscopy; 2) measuring GABA release within the rNA by using microdialysis; and 3) performing physiological experiments to determine the effects of selective blockade of GABA(A) receptors expressed by AVPNs in the rNA region on vl PAG-induced airway relaxation, thereby defining the role of the GABA(A) receptor subtype in this process. We observed that AVPNs located in the rNA region do express the GABA(A) receptor beta-subtype. In addition, we demonstrated that activation of vl PAG induced GABA release within the rNA region, and this release was associated with airway smooth muscle relaxation. Blockade of the GABA(A) receptor subtype expressed by AVPNs in the rNA by bicuculline diminished the inhibitory effects of vl PAG stimulation on airway smooth muscle tone. These data indicate, for the first time, that activation of vl PAG dilates the airways by a release of GABA and activation of GABA(A) receptors expressed by AVPNs.