Porcine Tracheal Smooth Muscle Research Articles

Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.

Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle

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.

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

The mitochondria and the sarcoplasmic reticulum (SR) are two major intracellular calcium-storing organelles that exhibit close functional interaction with each other. Close spatial association is believed to be important for their functional interaction. In this study, we have characterized the spatial relationship between the SR and the mitochondria in porcine tracheal smooth muscle cells (TSMC) under different conditions. By examining the cross-section of unstimulated TSMC with electron microscopy, we found that 99.4 ± 0.5% of the mitochondria seen on random cross-sections were situated within 30 nm of the SR and that 82.2 ± 6.7% of the mitochondria were completely enveloped by the SR network. Overall, 48.0 ± 3.5% of the mitochondrial outer membrane was within 30 nm with the SR. After stimulation of the TSMC with acetylcholine (ACh) or 80 mM [K +] solution 97.0 ± 2.1% and 98.6 ± 1.4% of the mitochondria observed were situated within 30 nm of the SR, respectively. However, the proportion of the mitochondria that was completely enveloped by the SR was significantly reduced to 12.2 ± 5.9% in ACh-stimulated cells and 9.7 ± 6.6% in 80 mM [K +] stimulated cells. The percentage of mitochondrial membrane closely associated with the SR was correspondingly lower at 10.1 ± 1.0% during ACh stimulation and 10.8 ± 0.9% during 80 mM [K +] stimulation. During smooth muscle cell stimulation, the SR appears to unwrap from the mitochondria and extend into the cytoplasm while maintaining close contact with the mitochondria over a smaller area. Such static and dynamic components of the close spatial association between the mitochondria and the SR may serve as a structural basis for the selective and efficient Ca 2+ trafficking between the two organelles in TSMC.

Regulation of Acetylcholine-Induced Phosphorylation of PLD1 in Porcine Tracheal Smooth Muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.

The bronchodilators 8- iso-prostaglandin E 2 and prostaglandin E 2 induce K + current suppression via thromboxane A 2 receptors in porcine tracheal smooth muscle

We examined relaxations and changes in K + current evoked by 8- iso-prostaglandin E 2 and prostaglandin E 2 in porcine tracheal smooth muscle. Both autacoids completely reversed cholinergic tone; blockade of thromboxane A 2 receptors had no effect on relaxations to either compound. 8- iso-prostaglandin E 2 and prostaglandin E 2 suppressed outward K + currents while the thromboxane A 2 receptor agonist U46619 (9, 11-dideoxy-9 a,11 a-methanoepoxy prostaglandin F 2α) had no significant effect. During thromboxane A 2 receptor antagonism, however, 8- iso-prostaglandin E 2 markedly augmented K + currents while prostaglandin E 2 no longer suppressed K + currents, indicating that the inhibition of K + currents by both compounds was thromboxane A 2 receptor-mediated. Furthermore, the observation that K + currents were augmented by 8- iso-prostaglandin E 2 but not by prostaglandin E 2 suggests that the salutory effect is not exerted through a prostaglandin E receptor. Additionally, our observations argue against any causal role for K + current activation in mediating relaxations evoked by isoprostanes or by prostaglandin E 2. We conclude that 8- iso-prostaglandin E 2 relaxes porcine tracheal smooth muscle independent of K + current activity, and that 8- iso-prostaglandin E 2 may also act at a non-thromboxane A 2/non-prostaglandin E receptor to augment K + currents.

Anesthetics inhibit acetylcholine-promoted guanine nucleotide exchange of heterotrimeric G proteins of airway smooth muscle.

Anesthetics inhibit airway smooth muscle contraction in part by a direct effect on the smooth muscle cell. This study tested the hypothesis that the anesthetics halothane and hexanol, which both relax airway smooth muscle in vitro, inhibit acetylcholine-promoted nucleotide exchange at the alpha subunit of the Gq/11 heterotrimeric G protein (Galphaq/11; i.e., they inhibit muscarinic receptor-Galphaq/11 coupling). The effect of halothane (0.38 +/- 0.02 mm) and hexanol (10 mm) on basal and acetylcholine-stimulated Galphaq/11 guanosine nucleotide exchange was determined in membranes prepared from porcine tracheal smooth muscle. The nonhydrolyzable, radioactive form of guanosine-5'-triphosphate, [S]GTPgammaS, was used as the reporter for Galphaq/11 subunit dissociation from the membrane to soluble fraction, which was immunoprecipitated with rabbit polyclonal anti-Galphaq/11 antiserum. Acetylcholine caused a significant time- and concentration-dependent increase in the magnitude of Galphaq/11 nucleotide exchange compared with basal values (i.e., without acetylcholine), reaching a maximal difference at 100 microm (35.9 +/-2.9 vs. 9.8 +/-1.2 fmol/mg protein, respectively). Whereas neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, both halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange produced by 30 microm acetylcholine (by 59% and 68%, respectively). Halothane and hexanol interact with the receptor-heterotrimeric G-protein complex in a manner that prevents acetylcholine-promoted exchange of guanosine-5(')-triphosphate for guanosine-5'-diphosphate at Galphaq/11. These data are consistent with the ability of anesthetics to interfere with cellular processes mediated by heterotrimeric G proteins in many cells, including effects on muscarinic receptor-G-protein regulation of airway smooth muscle contraction.

Inhibition of smooth muscle contraction by magnolol and honokiol in porcine trachea.

Magnolol and honokiol are the two major phenolic constituents of the plant medicine "Houpo" ( Magnolia obovata), which is used in the treatment of chest tightness and asthma. The aim of this study was to investigate the influence of magnolol and honokiol on smooth muscle tone in porcine trachea. Magnolol and honokiol (0.1 - 100 microM) inhibited carbachol- and high K +-induced muscle contractions in a concentration-dependent fashion, but did not affect basal muscle tension. After washout of these pretreatments, carbachol- and high K +-evoked muscle contractions were still abolished, suggesting that the inhibition was irreversible. Magnolol and honokiol also concentration-dependently decreased the Ca 2+-dependent muscle contraction induced by high K + depolarization. Ca 2+ channel antagonists attenuated carbachol-induced muscular response by approximately 30 %, but did not further potentiate the inhibitory actions of magnolol and honokiol on muscle contraction. However, the inhibitory effects of magnolol and honokiol on carbachol-evoked muscular contractile response were partially reversed after removal of Ca 2+ channel antagonist pretreatment. Alternatively, caffeine-elicited muscle contractions were not altered by magnolol, honokiol, and verapamil. In conclusion, the relaxant effects of magnolol and honokiol on porcine tracheal smooth muscle suggest an association with the blockade of Ca 2+ influx through voltage-operated Ca 2+ channels instead of Ca 2+ release from intracellular Ca 2+ stores. The magnolol- and honokiol-induced inhibitions on tracheal smooth muscle contraction may be relevant to the claimed therapeutic effects of the extract from magnolia bark and contribute to their pharmacological effects by acting as anti-asthmatic agents.

Calcium-independent contraction and sensitization of airway smooth muscle by p21-activated protein kinase.

In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce Ca(2+)-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433-23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces Ca(2+)-independent contractions and/or Ca(2+) sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of Ca(2+) (pCa > 9), constitutively active glutathione-S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to approximately 40% of the maximal force generated by Ca(2+) at pCa 4.4. In addition, GST-mPAK3 enhanced Ca(2+) sensitivity of contraction by increasing force generation by 80% at intermediate Ca(2+) concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK3(K297R) had no effect on force production. Using antibody against one of the PAK-phosphorylated sites (Ser(657)) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes Ca(2+)-independent contractions and produces Ca(2+) sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation.

The Repolarizing Effects of Volatile Anesthetics on Porcine Tracheal and Bronchial Smooth Muscle Cells

The Repolarizing Effects of Volatile Anesthetics on Porcine Tracheal and Bronchial Smooth Muscle Cells

Inhibitory effects of volatile anesthetics on K+ and Cl- channel currents in porcine tracheal and bronchial smooth muscle.

K+ and Ca2+-activated Cl- (ClCa) channel currents have been shown to contribute to the alteration of membrane electrical activity in airway smooth muscle. This study was conducted to investigate the effects of volatile anesthetics, which are potent bronchodilators, on the activities of these channels in porcine tracheal and bronchial smooth muscles. Whole-cell patch clamp recording techniques were used to investigate the effects of superfused isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) on K+ and ClCa channel currents in dispersed smooth muscle cells. Isoflurane and sevoflurane inhibited whole-cell K+ currents to a greater degree in tracheal versus bronchial smooth muscle cells. More than 60% of the total K+ currents in tracheal smooth muscle appeared to be mediated through delayed rectifier K+ channels compared with less than 40% in bronchial smooth muscle. The inhibitory effects of the anesthetics were greater on the delayed rectifier K+ channels than on the remaining K+ channels. Cl- currents through ClCa channels were significantly inhibited by the anesthetics. The inhibitory potencies of the anesthetics on the ClCa channels were not different in tracheal and bronchial smooth muscle cells. Volatile anesthetics isoflurane and sevoflurane significantly inhibited Cl- currents through ClCa channels, and the inhibitory effect is consistent with the relaxant effect of volatile anesthetics in airway smooth muscle. Different distributions and different anesthetic sensitivities of K+ channel subtypes could play a role in the different inhibitory effects of the anesthetics on tracheal and bronchial smooth muscle contractions.

The repolarizing effects of volatile anesthetics on porcine tracheal and bronchial smooth muscle cells.

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 Repolarizing Effects of Volatile Anesthetics on Porcine Tracheal and Bronchial Smooth Muscle Cells

The Repolarizing Effects of Volatile Anesthetics on Porcine Tracheal and Bronchial Smooth Muscle Cells

Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation

Muscarinic receptor agonists transiently activate phospholipase D in tracheal smooth muscle. Muscarinic activation of phospholipase D in this tissue is dependent on activation of protein kinase C and an unidentified pathway that is not protein kinase C dependent. Cholinergic agents have also been shown to activate phospholipase D by pathways linked to the small G protein, RhoA. This study explores the relationship between muscarinic activation of phophatidylinositol 3-kinase and activation of RhoA, and examines whether phospholipase D activation is dependent on either pathway in tracheal smooth muscle. Wortmannin or 2-(4-morphonyl)-8-phenyl-4 H-1-benzopyran-4-one (LY-294002), putative specific inhibitors of phophatidylinositol 3-kinase, significantly inhibit acetylcholine-induced formation of phosphatidylethanol and also block acetylcholine-induced translocation of RhoA to the membrane. In previous experiments calphostin C, a protein kinase C inhibitor, partially inhibited both acetylcholine-induced and phorbol-12-myristate-13-acetate (PMA)-induced phosphatidylethanol formation. In the present study calphostin C did not block acetylcholine-induced RhoA translocation to the membrane. However, the Rho kinase inhibitor, N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), significantly inhibited acetylcholine-induced phosphatidylethanol formation, but had no effect on activation of phospholipase D by PMA. Acetylcholine treatment also stimulated the phosphorylation of the 110-kDa subunit of phosphatidylinositol 3-kinase. Phosphorylation of phosphatidylinositol 3-kinase 110-kDa subunit could be blocked by wortmannin in a concentration-dependent manner, and acetylcholine-induced phosphatidylinositol 3-kinase activity was significantly inhibited by wortmannin. LY-294002 also inhibited acetylcholine-induced phosphorylation of 110-kDa subunit and activation of phosphatidylinositol 3-kinase. These results suggest that acetylcholine stimulation translocates RhoA to the membrane by a phosphatidylinositol 3-kinase-dependent mechanism and acetylcholine-induced phospholipase D stimulation is at least partly mediated via phosphatidylinositol 3-kinase, however, protein kinase C appears to activate phospholipase D independent of phosphatidylinositol 3-kinase or RhoA activation in porcine tracheal smooth muscle.

Different Inhibitory Effects of Volatile Anesthetics on T- and L-type Voltage-dependent Ca2+Channels in Porcine Tracheal and Bronchial Smooth Muscles

The distal airway is more important in the regulation of airflow resistance than is the proximal airway, and volatile anesthetics have a greater inhibitory effect on distal airway muscle tone. The authors investigated the different reactivities of airway smooth muscles to volatile anesthetics by measuring porcine tracheal or bronchial (third to fifth generation) smooth muscle tension and intracellular concentration of free Ca2+ ([Ca2+]i) and by measuring inward Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDCs). Intracellular concentration of free Ca2+ was monitored by the 500-nm light emission ratio of Ca2+ indicator fura-2. Isometric tension was measured simultaneously. Whole-cell patch clamp recording techniques were used to investigate the effects of volatile anesthetics on ICa in dispersed smooth muscle cells. Isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) was introduced into a bath solution. The volatile anesthetics tested had greater inhibitory effects on carbachol-induced bronchial smooth muscle contraction than on tracheal smooth muscle contraction. These inhibitory effects by the anesthetics on muscle tension were parallel to the inhibitory effects on [Ca2+]i. Although tracheal smooth muscle cells had only L-type VDCs, some bronchial smooth muscle cells (approximately 30%) included T-type VDC. Each of the two anesthetics significantly inhibited the activities of both types of VDCs in a dose-dependent manner; however, the anesthetics had greater inhibitory effects on T-type VDC activity in bronchial smooth muscle. The existence of the T-type VDC in bronchial smooth muscle and the high sensitivity of this channel to volatile anesthetics seem to be, at least in part, responsible for the different reactivities to the anesthetics in tracheal and bronchial smooth muscles.

Leukotriene C(4) enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway.

1. An unsaturated fatty acid, leukotriene C(4) (LTC(4)), has a potent contractile effect on human airway smooth muscle, and has been implicated in the pathogenesis of human asthma. Using front-surface fluorometry with fura-PE3, the effect of LTC(4) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were investigated in porcine tracheal smooth muscle strips. 2. The application of LTC(4) induced little or no contraction despite a small and transient increase in [Ca(2+)](i). In the presence of LTC(4), however, the contractions evoked by high K(+) depolarization or a low concentration of carbachol (CCh) were markedly enhanced without inducing any changes in the [Ca(2+)](i) levels, thus indicating that LTC(4) increases the Ca(2+) responsiveness of the contractile apparatus. This LTC(4)-induced increase in Ca(2+) responsiveness could partly be reproduced in the permeabilized preparation of tracheal smooth muscle strips. 3. The LTC(4)-induced enhancement of contraction was accompanied by an increase in myosin light chain (MLC) phosphorylation and was blocked by a rho kinase inhibitor (Y-27632), but not by either a PKC inhibitor (calphostin C) or a tyrosine kinase inhibitor (genistein). 4. These results indicated that, in porcine tracheal smooth muscle, LTC(4) enhances the contraction by increasing the Ca(2+) responsiveness of the contractile apparatus in a MLC phosphorylation dependent manner, possibly through the activation of the rho-rho kinase pathway.

Interaction Between Volatile Anesthetics and Hypoxia in Porcine Tracheal Smooth Muscle

We investigated the direct interaction between the volatile anesthetics, isoflurane and sevoflurane, and hypoxia in porcine tracheal smooth muscle in vitro by simultaneously measuring muscle tension and intracellular concentration of free Ca(2+) ([Ca2+]i). Muscle tension was measured by using an isometric transducer, and [Ca2+]i was measured by using fura-2, an indicator of Ca2+. Under the condition of bubbling with 95% O2/5% CO2, [Ca2+]i was increased by 1 microM carbachol with a concomitant contraction. Volatile anesthetics significantly inhibited both carbachol-induced muscle contraction and increase in [Ca2+]i. Hypoxia bubbled with 95% N(2)/5% CO2 inhibited the muscle contraction by 30% with an increase in [Ca2+]i by 20%. Exposure to hypoxia substantially enhanced the inhibitory effects of these anesthetics on carbachol-induced muscle contraction, whereas the decreases in [Ca2+]i were significantly prevented by hypoxia. Under Ca2+-free conditions, hypoxia significantly decreased the muscle contraction by 20%; however, it still increased [Ca2+]i by 15%. Exposure to the anesthetics significantly enhanced the inhibitory effect of hypoxia on the muscle contraction; however, it appeared to have little effect on [Ca2+]i. Hypoxia inhibits airway smooth muscle contraction independently of intracellular Ca2+, and it substantially potentiates the inhibitory effects of volatile anesthetics on airway smooth muscle contraction. Hypoxia inhibits agonist-induced tracheal smooth muscle contraction with an increase in free Ca2+ [Ca2+]i, which comes from intracellular Ca2+ stores. Hypoxia also potentiates the inhibitory effect of volatile anesthetics on airway smooth muscle contraction. Conversely, there is a possibility that the treatment of asthmatic patients with oxygen partially attenuates the inhibitory effect of volatile anesthetics on airway smooth muscle contractility.

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of β-NAD + to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5′-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS–PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K m values of 30.4±1.5 and 695.3±171.2 μM and V max values of 330.4±90 and 102.8±17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca 2+] i in porcine TSM cells.

Room G, 10/17/2000 2: 00 PM - 4: 00 PM (PS) Interaction between Volatile Anesthetics and Hypoxia in Porcine Tracheal Smooth Muscle

<b>Room G, 10/17/2000 2: 00 PM - 4: 00 PM (PS) Interaction between Volatile Anesthetics and Hypoxia in Porcine Tracheal Smooth Muscle</b>

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

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (~1.5 μm3) and global (whole cell) changes in [Ca2+]iwere measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]ioscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]ioscillations were ‘biphasic’ with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]ioscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]ioscillations lies in the establishment of mean [Ca2+]ilevel for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.