Porcine Tracheal Smooth Muscle Research Articles

Spatial and temporal aspects of calcium sparks in porcine tracheal smooth muscle cells.

Spontaneous, localized intracellular Ca(2+) concentration ([Ca(2+)](i)) transients (Ca(2+) sparks) in skeletal, cardiac, and smooth muscle cells are thought to represent Ca(2+) release through ryanodine-receptor (RyR) channels. In porcine tracheal smooth muscle (TSM) cells, ACh induces propagating [Ca(2+)](i) oscillations that also represent Ca(2+) release through RyR channels. We used real-time confocal imaging to examine the spatial and temporal relationships of Ca(2+) sparks to propagating [Ca(2+)](i) oscillations in TSM cells. Ca(2+) sparks within an intracellular region displayed different spatial Ca(2+) distributions with every occurrence. The amplitudes of Ca(2+) sparks within a region were approximately integer multiples of the smallest response. However, across different regions, the attributes of Ca(2+) sparks varied considerably. Individual sparks were often grouped together and coupled across adjacent regions. Fusion of individual sparks produced large local elevations in [Ca(2+)](i) that occasionally triggered a propagating [Ca(2+)](i) wave. The incidence of sparks was increased by ryanodine and caffeine but was unaffected by removal of extracellular Ca(2+). Exposure to ACh triggered repetitive, propagating [Ca(2+)](i) oscillations that always originated from foci with a high spark incidence. The [Ca(2+)](i) oscillations disappeared with the removal of ACh, and Ca(2+) sparks reappeared. We conclude that agonist-induced [Ca(2+)](i) oscillations represent a spatial and temporal integration of local Ca(2+)-release events through RyR channels in TSM cells.

Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity.

Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [3H]phosphatidic acid ([3H]PA) and [3H]phosphatidylethanol ([3H]PEth) after labeling the muscle strips with [3H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [3H]PA and [3H]PEth in a concentration-dependent manner (>105-400% vs. controls in the presence of 10(-6) to 10(-4) M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10(-6) M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10(-8) to 10(-6) M) and GFX (10(-8) to 10(-6) M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

P–573 - Mechanism for the leukotriene C4-induced enhancement of contraction in porcine tracheal smooth muscle.

P–573 - Mechanism for the leukotriene C4-induced enhancement of contraction in porcine tracheal smooth muscle.

Role of M 2 muscarinic receptors in airway smooth muscle contraction

Airway smooth muscle expresses both M 2 and M 3 muscarinic receptors with the majority of the receptors of the M 2 subtype. Activation of M 3 receptors, which couple to G q, initiates contraction of airway smooth muscle while activation of M 2 receptors, which couple to G i, inhibits β-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca 2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca 2+ sensitization by acetylcholine (ACh) and endothelin- 1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca 2+] in strips permeabilized with Staphylococcal α-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca 2+]. Pretreatment with pertussis toxin for 18–20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPγS. We conclude that the M 2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca 2+] via the heterotrimeric G-protein G i.

Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells.

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]i when Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In beta-escin-permeabilized cells, heparin inhibition of inositol 1,4, 5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]i oscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in approximately 20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]i oscillations, inhibiting oscillations in approximately 70% of cells at 2 MAC. The fall time of [Ca2+]i oscillations and the rate of fall of the [Ca2+]i response to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]i oscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4, 5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.

Molecular signaling mechanisms involved in Ach-induced cell proliferation in porcine tracheal smooth muscle cells

Molecular signaling mechanisms involved in Ach-induced cell proliferation in porcine tracheal smooth muscle cells

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.

Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle.

The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with beta-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM-10 microM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 microM) and by the RyR blockers ruthenium red (10 microM) and ryanodine (10 microM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]i oscillations induced by acetylcholine (ACh), addition of 100 nM and 1 microM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]i oscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]i response to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]i oscillations.

The role of cGMP in the relaxation to nitric oxide donors in airway smooth muscle

The aim of this study was to determine the effect of the soluble guanylyl cyclase inhibitors methylene blue and LY83583 (6-anilino-5,8-quinolinedione) on relaxation and increases in intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) concentration ([cGMP] i) induced by sodium nitroprusside, 3-morpholinosydnonimine (SIN-1) and diethylamine–nitric oxide (NO) in porcine tracheal smooth muscle in vitro. We measured (1) the effect of NO donors on isometric force and [cGMP] i and (2) the ability of methylene blue and LY83583 to antagonize these effects. In muscle strips contracted with carbachol (0.1–0.3 μM), both sodium nitroprusside and diethylamine–NO caused relaxation and an increase in [cGMP] i. By contrast, SIN-1 caused a relaxation which was not associated with a concomitant increase in [cGMP] i. Methylene blue (10 μM) and LY83583 (10 μM) completely blocked the increase in [cGMP] i induced by sodium nitroprusside and diethylamine–NO; however substantial relaxation remained. It is concluded that in porcine airway smooth muscle, (1) relaxation induced by some NO donors may occur without a concomitant increase in [cGMP] i; and (2) whereas relaxation induced by some NO donors may be associated with increases in [cGMP] i, the relaxation is not completely dependent upon it.

Comparative study of effects of isoproterenol and vasoactive intestinal polypeptide on voltage-dependent Ca 2+ and Ca 2+-activated K + currents in porcine tracheal smooth muscle cells

Effects of isoproterenol (Iso) and vasoactive intestinal polypeptide (VIP) on voltage-dependent Ca 2+ channel ( I Ca ) and Ca 2+-activated K + channel current ( I K- Ca ) in porcine tracheal smooth muscle cells were examined. When K + currents were inhibited using a Cs-rich pipette solution, application of 0.1–1 μM Iso or 1–10 nM VIP increased I Ca by 20–30%. On the other hand, I K- Ca elicited upon depolarization and spontaneous transient outward K+ currents (STOCs) recorded at a holding potential of -50 mV were enhanced by 80–100% in the presence of 0.1 μM Iso or 1 nM VIP.

Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells

Intracellular Ca2+ ([Ca2+]i) regulation in smooth muscle involves multiple mechanisms such as second messengers and ion channels. Intra- and inter-cellular heterogeneities in these mechanisms are likely, and will be reflected by heterogeneities in [Ca2+]i. In the present study, real-time confocal imaging was used to examine intracellular and intercellular heterogeneity in spontaneous Ca2+ sparks and acetylcholine-induced [Ca2+]i oscillations in porcine tracheal smooth muscle (TSM) cells. Ca2+ sparks were highly localized to multiple (2-5) foci in a cell. Individual sparks displayed relatively constant rise times (14.5 +/- 0.3% variance) and amplitudes (11.1 +/- 0.2% variance), but across regions these attributes varied. The incidence of sparks was often coupled across adjacent regions (r2 = 0.93 +/- 0.04). Spark frequency was increased approximately 350% by ryanodine and caffeine, suggesting that they represent unitary Ca2+ release through ryanodine receptor (RyR) channels. In TSM cells, acetylcholine induced [Ca2+]i oscillations that initiated from foci with the highest spark frequency. Results using beta-escin-permeabilized TSM cells indicated that [Ca2+]i oscillations also represent Ca2+ release through RyR channels. [Ca2+]i oscillations displayed intracellular heterogeneity in amplitude (30 +/- 4% variance) and intercellular heterogeneities in amplitude (100-800 nM) and frequency (5-35 per minute). Within a region, the amplitude and frequency of [Ca2+]i oscillations were correlated to both acetylcholine concentration (r = -0.79 +/- 0.04 for amplitude and 0.77 +/- 0.05 for frequency) and basal [Ca2+]i level (r = -0.94 +/- 0.02 for amplitude and 0.84 +/- 0.03 for frequency). Compared with TSM cells, acetylcholine-induced [Ca2+]i oscillations in bronchial cells were slower and lower in amplitude. We conclude that intracellular and intercellular heterogeneity in [Ca2+]i levels in airway smooth muscle reflects heterogeneities in Ca2+ regulatory mechanisms.

Inhibition of voltage-dependent Ca 2+ channels of porcine tracheal smooth muscle by the novel Ca 2+ channel antagonist RWJ-22108

1. 1. We compared electrophysiological effects of the bronchoselective Ca 2+ channel antagonist RWJ-22108 on voltage-dependent Ca 2+ channels (VDCs) of porcine tracheal smooth muscle cells to the effects of nicardipine and verapamil. 2. 2. Each of the three Ca 2+ channel antagonists tested inhibited inward Ca 2+ currents ( I Ca ) measured by whole-cell patch clamp techniques. Inhibition was dose-dependent with ∼50% inhibition of peak I Ca at +20 mV obtained with 3 × 10 −6M RWJ-22108, 3 × 10 −7M nicardipine, or 10 −5M verapamil. 3. 3. Both RWJ-22108 (3 × 10 −6M) and nicardipine (3 × 10 −7M) shifted the voltage dependence of steady-state inactivation to more negative potentials; however, the change in the potential of half-maximal inactivation induced by RWJ-22108 (−18 mV) was significantly greater than that induced by nicardipine (−12 mV). Verapamil did not alter the voltage dependence of inactivation. 4. 4. We conclude that inhibition of VDCs by RWJ-22108 is qualitatively similar to that by nicardipine but with a greater stabilizing effect on the inactivated channel state.

Role of ryanodine receptor channels in Ca2+ oscillations of porcine tracheal smooth muscle.

Acetylcholine (ACh) induces repetitive, propagating intracellular Ca2+ concentration ([Ca2+]i) oscillations in porcine tracheal smooth muscle (TSM) cells. Using real-time confocal microscopy, we examined the role of sarcoplasmic reticulum (SR) Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine receptor (RyR) channels in ACh-induced [Ca2+]i oscillations. In beta-escin permeabilized TSM cells, exposure to ACh in the presence of GTP also resulted in [Ca2+]i oscillations. [Ca2+]i oscillations could not be initiated by IP3 alone; however, an elevation of [Ca2+]i was observed. During ongoing [Ca2+]i oscillations, exposure to heparin, an IP3 receptor antagonist, caused a slowing of oscillation frequency but not complete inhibition. In contrast, ruthenium red, a RyR antagonist, completely abolished ACh-induced [Ca2+]i oscillations. Reverse transcriptase-polymerase chain reaction of TSM mRNA demonstrated the expression of RyR-2 and RyR-3 isoforms of the RyR. These results indicate that SR Ca2+ release through RyR channels is critical for ACh-induced [Ca2+]i oscillations in porcine TSM cells.

Nitric oxide inhibits ACh-induced intracellular calcium oscillations in porcine tracheal smooth muscle.

With real-time confocal microscopy, the effect of three nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and diethylamine NO adduct, on the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh) was examined. ACh initiated propagating [Ca2+]i oscillations in TSM cells, which were inhibited by NO donors. 8-Bromoguanosine 3',5'-cyclic monophosphate slowed the frequency of [Ca2+]i oscillations but did not completely inhibit oscillations, suggesting that the effects of NO donors are only partially mediated via guanosine 3',5'-cyclic monophosphate-dependent mechanisms. After preexposure to NO donors, ACh induced a small biphasic [Ca2+]i response that was blocked by nifedipine, suggesting a lack of effect on Ca2+ influx through voltage-gated channels. In addition, NO donors did not inhibit Ca2+ influx induced by BAY K 8644. The [Ca2+]i response to caffeine was inhibited by NO donors, indicating inhibition of sarcoplasmic reticulum (SR) Ca2+ release. When Ca2+ influx and SR Ca2+ reuptake were blocked, basal [Ca2+]i increased, and this was inhibited by NO donors, suggesting enhanced Ca2+ efflux. These results indicate that NO donors inhibit [Ca2+]i oscillations by blocking SR Ca2+ release and enhancing Ca2+ extrusion.

NITRIC OXIDE (NO) INHIBITS [Ca2+]i RESPONSE TO ACETYLCHOLINE BY REGULATING SECOND MESSENGER PRODUCTION IN AIRWAY SMOOTH MUSCLE. † 1802

NO inhibits acetylcholine (ACh)-induced intracellular Ca2+([Ca2+]i) oscillations in b-escin permeabilized porcine tracheal smooth muscle (TSM) cells. Using real-time confocal microscopy and radioimmunoassay, we determined the mechanisms by which NO inhibits the [Ca2+]i response to ACh. Heparin, a selective IP3 receptor antagonist, and U73122, a phospholipase C inhibitor, slowed the frequency of ongoing ACh-induced oscillations. Pre-exposure to U73122 or heparin prevented the initiation of ACh-induced [Ca2+]i oscillations. The [Ca2+]i response to ACh was abolished by ruthenium red, a ryanodine receptor (RyR) antagonist, and by 8-amino-cyclic ADP-ribose, an antagonist of cyclic ADP-ribose (cADPR) receptor. The [Ca2+]i response to ACh, IP3 and cADPR were abolished by s-nitroso-n-acetylpenicillamine (SNAP), a NO donor. SNAP reduced ACh-induced elevation of IP3 and cADPR levels. These results indicate that ACh-induced [Ca2+]i oscillations involve Ca2+ release by IP3 and cADPR and NO regulates their production. Supported by Graduate School, Univ. of Minnesota, NIH grant HL51736 and Abbott Laboratories.

Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells.

Using real-time confocal microscopy, we examined the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh). Exposure to ACh caused regenerative, propagating [Ca2+]i oscillations. The amplitude and fall time of the [Ca2+]i oscillations were inversely correlated to basal [Ca2+]i, whereas the frequency and rise time were directly correlated to basal [Ca2+]i. ACh-induced [Ca2+]i oscillations were initiated in the absence of extracellular Ca2+ and after membrane depolarization with KCl, suggesting that 1) [Ca2+]i oscillations primarily arise by release from internal stores such as the sarcoplasmic reticulum (SR), and 2) Ca2+ influx is necessary for maintenance of oscillations. Exposure to both caffeine and ryanodine inhibited ongoing ACh-induced [Ca2+]i oscillations, suggesting a role for caffeine-sensitive ryanodine receptor (RyR) SR Ca2+ channels. Inhibition of SR Ca2+ reuptake by thapsigargin increased basal [Ca2+]i and decreased [Ca2+]i oscillation amplitude, suggesting that Ca2+ reuptake is also essential. The present results suggest that [Ca2+]i oscillations in porcine TSM cells involve repetitive Ca2+ release and reuptake from RyR channels, perhaps through a Ca2+ -induced Ca2+ release mechanism.

Relaxation of porcine tracheal smooth muscle by parathyroid hormone-related protein

Parathyroid hormone-related protein (PTHrp) has been shown to relax uterine and gastrointestinal smooth muscles, but the mechanisms underlying its effects have not been characterized. Furthermore, its effect on pulmonary smooth muscle is unknown. Therefore we designed the present study to determine the PTHrp dose-response; the interaction of PTHrp and PTH; and the role of cyclic nucleotides and potassium channels in the PTHrp response in porcine tracheal smooth muscle (TSM). Our results indicate that, (1-34)PTHrp causes dose-dependent relaxation of TSM; that (1-34)PTHrp and (1-34)PTH demonstrate cross-tachyphylaxis to one another; that phosphodiesterase inhibition augments and phosphodiesterase stimulation attenuates the relaxation response while guanylate cyclase blockade has little effect, and that charybdotoxin and iberiotoxin, inhibitors of large conductance, Ca2+-activated, K+ channels, diminish the relaxation response. These findings suggest that (1-34)PTHrp-induced relaxation of TSM is mediated through a common PTHrp/PTH pathway or receptor, stimulation of cAMP and activation of large conductance, Ca2+-activated, K+ channels.

Nitric oxide inhibits calcium release from sarcoplasmic reticulum of porcine tracheal smooth muscle cells.

In the present study, effects of the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), on sarcoplasmic reticulum (SR) Ca2+ release were examined in freshly dissociated porcine tracheal smooth muscle (TSM) cells. Fura 2-loaded TSM cells were imaged using video fluorescence microscopy. SR Ca2+ release was induced by acetylcholine (ACh), which acts principally through inositol 1,4,5-trisphosphate (IP3) receptors, and by caffeine, which acts principally through ryanodine receptors (RyR). SNAP inhibited ACh-induced SR Ca2+ release at both 0 and 2.5 mM extracellular Ca2+. Degraded SNAP had no effect on ACh-induced SR Ca2+ release. SNAP also inhibited caffeine-induced SR Ca2+ release. ACh-induced Ca2+ influx was not affected by SNAP when SR reloading was blocked by thapsigargin. SNAP also did not affect SR Ca2+ reuptake. The membrane-permeant analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromo-cGMP, mimicked the effects of SNAP. These results suggest that, in porcine TSM cells, SNAP reduces the intracellular Ca2+ response to ACh and caffeine by inhibiting SR Ca2+ release through both IP3 and RyR, but not by inhibiting influx or repletion of the SR Ca2+ stores. These effects are likely mediated via cGMP-dependent mechanisms.

Halothane counteracts acetylcholine-induced increase in Ca 2+ sensitivity of the contractile apparatus in airway smooth muscle

The direct relaxing effect of halothane on airway smooth muscle has been reported to involve the reduction of the cytosolic Ca 2+ concentration ([Ca 2+] i) and the [Ca 2+] i-independent inhibitory mechanism. To clarify the extent of the contribution of these mechanisms, the effect of halothane on the [Ca 2+] i-tension relationship in porcine tracheal smooth muscle strips was evaluated, using fura-2 fluorometry. The control [Ca 2+] i-tension relationship was constructed from data of [Ca 2+] i and tension during the contractions induced by the stepwise increment of extracellular Ca 2+ concentration under high K + depolarization. In the presence of acetylcholine (1 μM), the [Ca 2+] i-tension relationship shifted upward, which indicated the acetylcholine-induced increase in the Ca 2+ sensitivity of the contractile apparatus. Halothane (0.034 mM), in the absence of acetylcholine, did not alter the increases in either [Ca 2+] i or tension, hence no change in the [Ca 2+] i-tension relationship. However, in the presence of acetylcholine, halothane did attenuate the acetylcholine-induced upward shift of the [Ca 2+] i-tension relationship. Halothane proved to have a potent attenuating effect on the acetylcholine-induced increase in Ca 2+ sensitivity of the contractile apparatus with little influence on [Ca 2+] i. This desensitization of the contractile apparatus to [Ca 2+] i may play a major role in the direct airway relaxing effect of halothane.

EFFECT OF SUPEROXIDE DISMUTASE INHIBITOR ON NITRIC OXIDE-INDUCED AIRWAY RELAXATION AND INTRACELLULAR Ca2+ TRANSIENTS. ▴ 2307

Endogenous production of superoxide anion (SA) is known to affect a variety of signal transduction pathways in smooth muscle. In this study, we investigated the effect of diethyldithiocarbamate (DETCA), an inhibitor of Zn, Cu-superoxide dismutase (SOD), on nitric oxide (NO)-mediated relaxation and intracellular Ca2- ([Ca2+]i) transients in porcine tracheal smooth muscle (TSM) cells. In carbachol (CH)-contracted TSM strips, DETCA (10 mM) inhibited relaxations mediated by cGMP and induced by NO released from intrinsic nerves as well as by the NO donor, sodium nitroprusside (SNP), but not that mediated by cAMP and induced by isoproterenol. In CH-contracted tissues, exposure to DETCA resulted in increased lucigenin-dependent chemiluminescence. DETCA did not inhibit the elevation of cGMP in response to SNP. In single, freshly isolated TSM cells loaded with fura-2/AM, exposure to 100 nM acetylcholine (ACh), which acts through the inositol 1,4,5-trisphosphate (IP3) receptor, resulted in elevation of [Ca2+]i. DETCA attenuated this Ca2+ response, but not to caffeine, an inducer of Ca2+ release through the ryanodine receptor. These results suggest that SA attenuates NO-mediated relaxations at a step distal to the generation of cGMP, and decreases ACh-induced elevation of [Ca2+]i by inhibiting IP3-induced Ca2+ release selectively. Supported by Univ. of Minnesota, Mayo Foundation, and NIH grant HL51736-02C. MSK was recipient of an Abbott Senior Fellowship.