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

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.

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.

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.

Nitric oxide (NO) causes tracheal smooth muscle (TSM) relaxation by inhibiting agonist-stimulated elevation of intracellular Ca2+([Ca2+]i). via cGMP-dependent mechanisms. In this study, we examined the effect of s-nitroso-n-acetylpenicillamine (SNAP), a NO donor, on the Ca2+ response to acetylcholine (ACh). Single, freshly isolated porcine TSM cells were loaded with fura-2/AM and [Ca2+]i was measured as the ratio of fura-2 emissions when excited at 340 and 380 nm, using a video fluorescence imaging system. SNAP (0.1-1.0 μM) inhibited the normal [Ca2+]i response to ACh (0.1-1.0 μM) at an extracellular Ca2+ ([Ca2+]o) of either 0 or 2.5 mM. After SNAP inhibition of [Ca2+]i response to ACh in 0 mM Ca2+, a large Ca2+ response was seen when [Ca2+]o was readjusted to 2.5 mM, indicating that Ca2+ influx was not affected by NO. After depleting the sarcoplasmic reticulum (SR) Ca2+ pool with ACh and re-exposing to 2.5 mM Ca2+ in the presence of SNAP, the subsequent ACh response in the absence of SNAP was unaffected, indicating that NO does not affect reloading of the SR. These results suggest that NO selectively affects SR Ca2+ release, but does not affect Ca2+ influx or SR reloading. Inhibition of SR Ca2+ release can lead to relaxation of TSM. Supported by NIH grants HL51736 and HL07111, Mayo Foundation, and Univ. of Minnesota. MSK was a recipient of an Abbott Senior Fellowship.

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.

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.

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.

While sympathetic stimulation of the heart produces chronotropic, inotropic, and lusitropic effects, increased frequency alone causes a positive force-frequency relationship (FFR) and frequency-dependent acceleration of relaxation (FDAR).1 That is, contraction amplitude and relaxation rate are increased with increasing frequency in most species (including humans). The key mechanism involved in the positive FFR is increased sarcoplasmic reticulum (SR) Ca2+ load, due to increased Ca2+ influx and decreased Ca2+ efflux.1,2 Ca2+ influx increases due to more L-type Ca2+ current ( I Ca) per unit time, while Ca2+ efflux via Na+-Ca2+ exchange (NCX) decreases because the diastolic time is reduced and [Na+]i increases. Enhanced SR Ca2+-pump function causes FDAR and also augments SR Ca2+ loading. Various signaling pathways are involved (eg, CaMKII).3 In human heart failure, the FFR reverses (ie, from positive to negative) due to an inability of the SR to increase Ca2+ content.4 This negative FFR is a main contributor to the loss of contractile reserve in the failing heart. Of many pathways that can modify FFR, nitric oxide (NO) signaling is the topic addressed by Khan et al in this issue of Circulation Research .5 NO synthase (NOS) produces NO from l-arginine, and cardiac myocytes express all three NOS isoforms.6,7 NOS1 (nNOS) and NOS3 (eNOS) are constitutively expressed and produce low amounts of NO (regulated by [Ca2+-calmodulin]i levels). NOS2 (iNOS) …

Spatial and temporal aspects of ACh-induced [Ca2+]ioscillations in porcine tracheal smooth muscle

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.

Stimulation of the tracheal muscle bundle by acetylcholine (ACh) results in the generation of asynchronous repetitive Ca2+ waves (ACW) in intact tracheal smooth muscle (TSM) cells. We showed previously that ACW underlie cholinergic excitation-contraction coupling in porcine TSM and that Ca2+ entry through the L-type voltage-gated Ca2+ channel (VGCC) contributes partially to maintenance of the ACW. However, the mechanism of the ACW remains undefined. In this study, we pharmacologically characterized the mechanism of ACh-induced ACW in the intact porcine tracheal muscle bundle. We found that inhibition of receptor-operated channels/store-operated channels (ROC/SOC) by SKF-96365 completely abolished the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Blockade of Na+/Ca2+ exchange with KB-R7943 or 2',4'-dichlorobenzamil or removal of extracellular Na+ resulted in nearly complete inhibition of the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid abolished the ongoing ACW. Application of 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C to inhibit the inositol 1,4,5-trisphosphate-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels produced no effect on ACh-mediated ACW and tonic contraction. However, pretreatment with caffeine or ryanodine inhibited ACh-induced ACW. Furthermore, application of procaine or tetracaine prevented the generation and abolished the ongoing ACh-mediated ACW and tonic contraction. Collectively, these results indicate that the ACh-stimulated ACW in porcine TSM are produced by repetitive cycles of Ca2+ release from SR through 2-APB- and xestospongin C-insensitive Ca2+ release channels, and plasmalemmal Ca2+ entry involving reverse-mode Na+/Ca2+ exchange, ROC/SOC, and L-type VGCC is required to refill the SR via SERCA to support the ongoing ACW.

This study was conducted to determine the effects of volatile anesthetics (potent bronchodilators) on membrane potentials in porcine tracheal and bronchial smooth muscle cells. We used a current-clamp technique to examine the effects of the volatile anesthetics isoflurane (1.5 minimum alveolar anesthetic concentration [MAC]) and sevoflurane (1.5 MAC) on membrane potentials of porcine tracheal and bronchial (third- to fifth-generation) smooth muscle cells depolarized by a muscarinic agonist, carbachol (1 microM). The effects of volatile anesthetics on muscarinic receptor binding affinity were also investigated by using a radiolabeled receptor assay technique. The volatile anesthetics isoflurane and sevoflurane induced significant repolarization of the depolarized cell membranes in the trachea (from -19.8 to -23.6 mV and to -24.8 mV, respectively) and bronchus (from -24.7 to -29.3 mV and -30.4 mV, respectively) without affecting carbachol binding affinity to the muscarinic receptor. The repolarizing effect was abolished by a Ca(2+)-activated Cl(-) channel blocker, niflumic acid. These results indicate that volatile anesthetic-induced repolarization of airway smooth muscle cell membranes might be caused by a change in Ca(2+)-activated Cl(-) channel activity and that the different repolarized effects of the volatile anesthetics could in part contribute to the different effects of volatile anesthetics on tracheal and bronchial smooth muscle contractions. By use of a current-clamp technique, the volatile anesthetics isoflurane and sevoflurane repolarized porcine airway smooth muscle cell membranes depolarized by a muscarinic agonist. This effect might be caused mainly by change in Ca(2+)-activated Cl(-) channel activity, not in K(+) channel activity.

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

Rearrangement of the close contact between the mitochondria and the sarcoplasmic reticulum in airway smooth muscle

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.