Porcine Tracheal Smooth Muscle Cells Research Articles

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.

MODULATION OF INTRACELLULAR Ca2+ TRANSIENTS BY NITRIC OXIDE IN AIRWAY SMOOTH MUSCLE CELLS. ▴ 2308

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.

Sodium nitroprusside stimulates Ca2+ -activated K+ channels in porcine tracheal smooth muscle cells.

To directly investigate the possible role of large-conductance Ca2+ -activated K+ (KCa) channels in nitro-vasodilator-induced relaxation of airway smooth muscle, we used cell-attached patch-clamp techniques to test the effects of sodium nitroprusside (SNP) on KCa channels in freshly dispersed porcine tracheal smooth muscle cells. Channel open-state probability (nPo) increased approximately 13-fold with exposure to 10(-5) M SNP, and this was partially reversed by addition of the guanylate cyclase inhibitors methylene blue (3 X 10(-4) M) or LY-83583 (5 X 10(-5) M). Pretreatment with the guanosine 3',5' -cyclic monophosphate (cGMP)-dependent protein kinase (G kinase) inhibitor Rp-8-(p-chlorophenylthio) cGMP-phosphorothioate (2 X 10(-5) M) prevented activation of KCa channels by SNP. We also tested the ability of G kinase to directly activate KCa channels in inside-out patches. G kinase (2.5 U/microliter) with ATP (0.5 mM) and cGMP (0.1 mM), but not ATP and cGMP alone, increased nPo approximately 23-fold. We conclude that SNP activates KCa channels in airway smooth muscle via guanylate cyclase and G kinase. Phosphorylation of the channel protein by G kinase may account for this response. Consequent membrane hyperpolarization and inhibition of Ca2+ entry through voltage-dependent channels may contribute to SNP-induced relaxation of airway smooth muscle.

P-229 - The role of endothelins in the porcine tracheal smooth muscle and epithelial cells.

P-229 - The role of endothelins in the porcine tracheal smooth muscle and epithelial cells.

Cholinergic regulation of voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells.

To investigate cholinergic regulation of voltage-dependent Ca2+ channels (VDCs) in airway smooth muscle, we measured inward currents through VDCs in enzymatically dispersed porcine tracheal smooth muscle cells using conventional (10 mM Ca2+ as charge carrier) and nystatin-perforated (5 mM Ba2+ as charge carrier) whole cell patch clamp techniques. Carbachol (CCh) had significant and dose-dependent inhibitory effects on inward currents (12% with 10(-7) M and 42% with 10(-6) M) in perforated whole cell clamp experiments, but had no effect on currents in conventional whole cell experiments. CCh also shifted the steady-state inactivation curve to more negative potentials. Further experiments tested the hypothesis that CCh inhibits VDCs in part by the activation of protein kinase C (PKC). Phorbol 12,13-diacetate, an exogenous PKC activator, inhibited currents through VDCs. and calphostin C, a specific PKC inhibitor, antagonized the inhibitory effect of CCh. Furthermore, intracellular exposure to the activating PKC fragment 530-558, using a pipette perfusion technique, also inhibited currents through VDCs. We conclude that cholinergic receptor stimulation can inhibit inward Ca2+ currents through VDCs of porcine tracheal smooth muscle and that this effect may be mediated in part by activation of PKC.

Arachidonic acid metabolites and glucocorticoid regulatory mechanism in cultured porcine tracheal smooth muscle cells.

To elucidate the signal transduction system in the production of prostaglandin E2 (PGE2) by porcine tracheal smooth muscle cells in culture (PTSMC), we examined the pattern of arachidonic acid metabolites released from PTSMC and the relationship between bradykinin-stimulated rises in intracellular calcium concentration ([Ca2+]i) and PGE2 production by PTSMC. We next examined the effect of dexamethasone on these parameters. Bradykinin induced a dose-dependent increase in both the rise in [Ca2+]i and PGE2 production by PTSMC. The increase in [Ca2+]i paralleled an increase in PGE2 production. High-performance liquid chromatography (HPLC) revealed that dexamethasone-treated PTSMC were suppressed to release arachidonic acid metabolites such as PGE2 and prostaglandin F2 alpha (PGF2 alpha). Incubation of PTSMC with 10(-6)M dexamethasone for 24 h significantly suppressed both the rise in [Ca2+]i and PGE2 production by PTSMC in response to bradykinin, and also significantly suppressed bradykinin-stimulated release of radioactivity from PTSMC prelabeled with 3H-labeled arachidonic acid (3H-AA). When PTSMC pretreated with dexamethasone were incubated with 170 nM prostaglandin H2 (PGH2) or 20 microM arachidonic acid; PTSMC synthesized less PGE2 than control PTSMC. Results suggest that bradykinin stimulates PTSMC to produce PGE2 via the signal transduction system including Ca2+, and dexamethasone appeared to suppress PGE2 production by reducing the activity of cytosolic phospholipase A2 (cPLA2) and PGE2 synthase. However, we failed to demonstrate the suppression of the activity of cyclooxygenase in PTSMC by dexamethasone. Since the elevation of [Ca2+]i is necessary for the contraction of airway smooth muscles, dexamethasone seems to reduce the contraction of airway smooth muscles by suppressing the rise in [Ca2+]i and the release of arachidonic acid metabolites. Reduced production of arachidonic acid metabolites may also contribute to improvement in the bronchial inflammation.

Intracellular pH regulates voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells

Changes in CO2 or in pH modify airway smooth muscle contractility. To investigate the mechanisms involved, we compared K(+)-induced contractions in porcine bronchial rings exposed to different CO2 concentrations and directly measured the effects of changes in intracellular (pHi) or extracellular pH (pHo) on Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDC) in porcine tracheal smooth muscle cells. Hypocapnia and hypercapnia caused leftward and rightward shifts, respectively, in the dose-response to K+ (P < 0.05) but did not change the maximum force obtained. Peak ICa (10 mM external Ca2+) elicited by depolarizing pulses from -80 mV was maximal [-265 +/- 12 pA (mean +/- SE), n = 19] at +10 mV. Intracellular acidification decreased the peak ICa at +10 mV from -261 +/- 20 pA to -177 +/- 12 pA (P < 0.05, n = 4), while intracellular alkalinization increased the peak ICa at +10 mV from -302 +/- 27 pA to -368 +/- 26 pA (P < 0.05, n = 4). Changes in pHo had little effect on ICa. There was no shift in the voltage-dependence of induced ICa with any change. We conclude that pHi, but not pHo, directly modulates the entry of Ca2+ into airway smooth muscle cells through VDC. This mechanism may contribute to regulation of airway tone by CO2.

Volatile anesthetics inhibit voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells

The relaxation of airway smooth muscle by volatile anesthetics is associated with a decreased concentration of intracellular free Ca2+. We hypothesized that inhibition of the entry of extracellular Ca2+ contributes to the relaxation. We therefore examined the effects of halothane, isoflurane, and sevoflurane on macroscopic voltage-activated Ca2+ currents (ICa) in porcine tracheal smooth muscle cells, using the whole cell patch-clamp technique. All three volatile anesthetics significantly inhibited ICa in a dose-dependent manner with no apparent shift in the voltage dependence of induced ICa. The order of inhibitory potencies for ICa was halothane > isoflurane > sevoflurane. When data were plotted as a function of the estimated anesthetic concentrations in the lipid phase, the potencies for inhibition of ICa by the three anesthetics were indistinguishable. We conclude that volatile anesthetics have an inhibitory effect on ICa of porcine tracheal smooth muscle cells at clinically relevant concentrations and that the inhibitory potencies of volatile anesthetics on ICa are closely related to their lipid-phase solubilities.

Inhibition by Halothane of Voltage-dependent Ca2+ Currents in Porcine Tracheal Smooth Muscle Cells

Inhibition by Halothane of Voltage-dependent Ca2+ Currents in Porcine Tracheal Smooth Muscle Cells

培養ブタ気管平滑筋細胞のアラキドン酸代謝に及ぼす小青竜湯と柴朴湯の作用

We studied the effect of Sho-seiryu-to and Saiboku-to on arachidonic acid metabolism by cultured porcine tracheal smooth muscle cells. HPLC analysis revealed that both cyclooxygenase derivatives, especially PGE2, and lipoxygenase derivatives released in the culture medium were increased during 24-hour incubation of the tracheal smooth muscle cells with 1, 000μg/ml of Sho-seiryu-to. In contrast, the incubation of the tracheal smooth muscle cells with 1, 000μg/ml of Saiboku-to decreased both cyclooxygenase and lipoxygenase derivatives released in the culture medium. Increased release of PGE2from tracheal smooth muscle cells incubated with Sho-seiryu-to may support the therapeutic benefit for bronchial asthma because of broncho-dilatation effect by PGE2, and the suppression of the production of arachidonic acid metabolites by Saiboku-to may show the antiinflammatory action of Saiboku-to.