Chloride and depolarization by acetylcholine in canine airway smooth muscle

1993 ◽  
Vol 71 (3-4) ◽  
pp. 284-292 ◽  
Author(s):  
E. E. Daniel ◽  
J. Jury ◽  
J.-P. Bourreau ◽  
L. Jager
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Chloride Channels ◽  
Reversal Potential ◽  
Chloride Ion ◽  
Resting Potential ◽  
Mechanical Responses ◽  
Intracellular Ca ◽  
Excitatory Junction Potentials ◽  
Sucrose Gap

The role of chloride channels has been examined in canine tracheal smooth muscle by recording mechanical responses to field stimulation and to acetylcholine (ACh) and by sucrose gap recording of excitatory junction potentials and ACh-induced electrical changes. The results of substitution studies using isethionate for chloride provided evidence that a chloride conductance contributes to the resting potential. The extrapolated reversal potential for ACh-induced depolarization was positive to the resting potential. Isethionate substitution inhibited ACh-induced depolarization, consistent with a contribution from increased Cl− conductance to the depolarization induced by ACh. However, closure of K+ channels and opening of a nonspecific cation channel could also contribute to depolarization. Further study of the effects of isethionate substitution during prolonged tissue exposure to chloride-free medium showed that retention or the accumulation of Ca2+ in intracellular stores was impaired. We conclude that effects of chloride deprivation on responses to ACh may reflect an early increase in Cl− conductance, but longer term changes reflect the requirement for this anion to maintain internal Ca2+ stores.Key words: chloride ion, airway smooth muscle, ion channels, release of intracellular Ca2+.

K+-channel blockers do not decrease acetylcholine depolarizations in canine trachealis

1992 ◽  
Vol 70 (1) ◽  
pp. 43-52 ◽  
Author(s):  
E. E. Daniel ◽  
J. Jury ◽  
R. Serio ◽  
L. P. Jager
Keyword(s):  
Chloride Channels ◽  
Reversal Potential ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
Resting Potential ◽  
K Channel ◽  
Channel Blockers ◽  
Membrane Effects ◽  
Sucrose Gap ◽  
Time Courses

Using the double sucrose gap, we have examined the role of K+ channels in the cholinergic depolarizations in response to field stimulation and acetylcholine (Ach) in canine trachealis. Acetylcholine-like depolarization per se decreased electrotonic potentials from hyperpolarizing currents. The net effect of acetylcholine (10−6 M) depolarization on membrane conductance was a small increase after the depolarization was compensated by current clamp. Reversal potentials for acetylcholine depolarization and for the excitatory junction potential (EJP) were determined by extrapolation to be 20–30 mV positive to the resting potential, previously shown to be approximately −55 mV. They were shifted positively by tetraethylammonium ion (TEA) at 20 mM or Ba2+ at 1 mM. TEA or Ba2+ initially depolarized the membrane and increased membrane resistance. Repolarization of the membrane restored any reductions in EJP amplitudes associated with depolarization. After 15 min, the membrane potential partially repolarized, and acetylcholine-induced depolarization and contractions were then increased by TEA. 4-Aminopyridine depolarized the membrane but decreased membrane resistance. Apamin (10−6 M), charybdotoxin (10−7 M), and glybenclamide (10−5 M) each failed to significantly depolarize membranes, increase membrane resistance, or reduce EJP amplitudes or depolarization to 10−6 M Ach. Glybenclamide reduced depolarizations to added acetylcholine slightly. TEA occasionally reduced the EJP markedly, but this was shown to be most likely a prejunctional effect mediated by norepinephrine release. TEA alone among K+-channel blockers slowed the onset and the time courses of the EJP as well as the acetylcholine-induced depolarization. K+-channel closure cannot be a complete explanation of acetylcholine-induced membrane effects on this tissue. Acetylcholine must have increased the conductance of an ion with a reversal potential positive to the resting potential in addition to any effect to close K+ channels.Key words: acetylcholine, tracheal smooth muscle, trachea, chloride channels, sucrose gap, potassium channels, tetraethylammonium, Ba2+.

Role of depolarization and calcium in contractions of canine trachealis from endogenous or exogenous acetylcholine

1991 ◽  
Vol 69 (4) ◽  
pp. 518-525 ◽  
Author(s):  
E. E. Daniel ◽  
J. Jury ◽  
R. Serio ◽  
L. P. Jager
Keyword(s):  
Chloride Channels ◽  
Membrane Resistance ◽  
Ca Channel ◽  
Field Stimulation ◽  
Mechanical Responses ◽  
Excitatory Junction Potential ◽  
Voltage Dependent ◽  
Excitatory Junction Potentials ◽  
Sucrose Gap ◽  
Junction Potential

The relationships of the electrical to the mechanical responses of the canine trachealis muscle during stimulation of its cholinergic nerves or exposure to exogenous acetylcholine were recorded in the single or the double sucrose gap. At 27 °C, the responses to a train of stimuli consisted of a transient depolarization excitatory junction potential of 10–30 mV followed by fading oscillations and contractions. When stimulus parameters were varied in the single sucrose gap, contractions were more closely associated with the occurrence of and varied in duration with the oscillations rather than with the amplitude of the EJP. Acetylcholine superfused at a concentration of 10−6 M for 30 s caused a prolonged depolarization of 10–20 mV, but a much larger contraction than could be elicited by nerve stimulation. None of the responses to acetylcholine was significantly affected by the Ca channel antagonists, nifedipine, nitrendipine, or verapamil in Ca channel blocking concentrations. When tissues were exposed to a Ca-free medium, the excitatory junction potentials and oscillations rapidly disappeared, but the electrical and mechanical responses to acetylcholine persisted and only gradually disappeared with repetitive exposures. Furthermore, in a medium with normal Ca2+ in the double sucrose gap, depolarization by 10–15 mV with an applied current caused no contraction, and repolarization to the normal membrane potential during acetylcholine-induced contraction caused no relaxation. Tetraethylammonium ion (20 mM) depolarized the membrane, increased membrane resistance, and enhanced the secondary oscillations and contractions after field stimulation. No other K+-channel blocker tested (Ba2+, apamin, 4-aminopyridine, glibenclamide, charybdotoxin) had the effect of prolonging secondary oscillations. We concluded that acetylcholine under our conditions acted to release internal Ca2+ and that the depolarization was secondary to that release or an associated event. Moreover, contractile responses to released acetylcholine during field stimulation do not appear to depend significantly on the opening of voltage-dependent Ca channels. The secondary oscillations were closely related to contraction; they seemed to be modulated by the opening of tetraethylammonium-sensitive, possibly Ca2+-activated K+ channels. The physiological functions, if any, of the initial depolarization associated with acetylcholine released (excitatory junction potential) or added remain unclear.Key words: acetylcholine, tracheal smooth muscle, trachea, chloride channels, sucrose gap, potassium channels.

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle

2005 ◽  
Vol 99 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Peter B. Helli ◽  
Evi Pertens ◽  
Luke J. Janssen
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Airway Smooth Muscle ◽  
Reversal Potential ◽  
Cyclopiazonic Acid ◽  
Membrane Potentials ◽  
Tracheal Smooth Muscle ◽  
Cation Channels ◽  
Cation Conductance ◽  
Intracellular Ca

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as “store-operated” cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an l-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl− currents, we found that CPA rapidly activated a membrane conductance ( ICPA) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of ICPA at physiological membrane potentials (i.e., −60 mV) that was accompanied by a shift in the reversal potential for ICPA toward more negative membrane potentials. In addition, ICPA was markedly inhibited by 10 μM Gd3+ and La3+ but was largely insensitive to 1 μM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.

scholarly journals Membrane Currents in Airway Smooth Muscle: Mechanisms and Therapeutic Implications

1997 ◽  
Vol 4 (1) ◽  
pp. 13-20
Author(s):  
Luke J Janssen
Keyword(s):  
Smooth Muscle ◽  
Ion Channels ◽  
Calcium Channels ◽  
Airway Smooth Muscle ◽  
Calcium Ion ◽  
Membrane Conductance ◽  
Chloride Ion ◽  
Potassium Ion Channels ◽  
Therapeutic Implications ◽  
Transient Activation

Electrophysiological and pharmacological techniques were used to characterize the membrane conductance changes underlying spasmogen-evoked depolarization in airway smooth muscle (ASM). Changes included a transient activation of chloride ion channels and prolonged suppression of potassium ion channels; both changes are triggered by release of internally sequestered calcium ion and in turn cause opening of voltage-dependent calcium channels. The resultant influx of calcium ions contributes to contraction as well as to refilling of the internal calcium ion pool. Bronchodilators, on the other hand, act in part through activation of potassium channels, with consequent closure of calcium channels. The tools used to study ion channels in ASM are described, and the investigations of the roles of ion channels in ASM physiology (autacoid-evoked depolarization and hyperpolarization) and pathophysiology (airway hyperresponsiveness) are summarized. Finally, how the relationship between ion channels and ASM function/dysfunction may relate to the treatment of asthma and related breathing disorders is discussed.

scholarly journals Airway smooth muscle CXCR3 ligand production: regulation by JAK-STAT1 and intracellular Ca2+

2013 ◽  
Vol 304 (11) ◽  
pp. L790-L802 ◽  
Author(s):  
X. Tan ◽  
Y. A. Alrashdan ◽  
H. Alkhouri ◽  
B. G. G. Oliver ◽  
C. L. Armour ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Immunoblot Analysis ◽  
Interferon Γ ◽  
Protein Activation ◽  
Early Activation ◽  
Plasmic Reticulum ◽  
Cxcr3 Ligand ◽  
Intracellular Ca ◽  
Jnk Activation

In asthma, airway smooth muscle (ASM) chemokine (C-X-C motif) receptor 3 (CXCR3) ligand production may attract mast cells or T lymphocytes to the ASM, where they can modulate ASM functions. In ASM cells (ASMCs) from people with or without asthma, we aimed to investigate JAK-STAT1, JNK, and Ca2+ involvement in chemokine (C-X-C motif) ligand (CXCL)10 and CXCL11 production stimulated by interferon-γ, IL-1β, and TNF-α combined (cytomix). Confluent, growth-arrested ASMC were treated with inhibitors for pan-JAK (pyridone-6), JAK2 (AG-490), JNK (SP-600125), or the sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) pump (thapsigargin), Ca2+ chelator (BAPTA-AM), or vehicle before and during cytomix stimulation for up to 24 h. Signaling protein activation as well as CXCL10/CXCL11 mRNA and protein production were examined using immunoblot analysis, real-time PCR, and ELISA, respectively. Cytomix-induced STAT1 activation was lower and CXCR3 ligand mRNA production was more sensitive to pyridone-6 and AG-490 in asthmatic than nonasthmatic ASMCs, but CXCL10/CXCL11 release was inhibited by the same proportion. Neither agent caused additional inhibition of release when used in combination with the JNK inhibitor SP-600125. Conversely, p65 NF-κB activation was higher in asthmatic than nonasthmatic ASMCs. BAPTA-AM abolished early CXCL10/CXCL11 mRNA production, whereas thapsigargin reduced it in asthmatic cells and inhibited CXCL10/CXCL11 release by both ASMC types. Despite these inhibitory effects, neither Ca2+ agent affected early activation of STAT1, JNK, or p65 NF-κB. In conclusion, intracellular Ca2+ regulated CXCL10/CXCL11 production but not early activation of the signaling molecules involved. In asthma, reduced ASM STAT1-JNK activation, increased NF-κB activation, and altered Ca2+ handling may contribute to rapid CXCR3 ligand production and enhanced inflammatory cell recruitment.

Electrophysiological differentiation of alpha-receptors on arteriolar smooth muscle

1983 ◽  
Vol 244 (4) ◽  
pp. H540-H545 ◽  
Author(s):  
K. G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Electrical Activity ◽  
Resting Potential ◽  
Vessel Size ◽  
Contraction Coupling ◽  
Alpha Receptors ◽  
Excitatory Junction Potentials ◽  
Coupling Mechanisms ◽  
Electrophysiological Differentiation

The effects of clonidine (a prototype of an alpha 2-agonist) and phenylephrine (a prototype of an alpha 1-agonist) on intracellularly recorded electrical activity and vessel size of feline submucosal arterioles were compared. Phenylephrine constricts the vessels and causes a depolarization and the initiation of oscillations of the membrane potential. These oscillations occasionally give rise to spike potentials. In contrast, clonidine produces no significant depolarization of the resting potential in spite of the simultaneous initiation of contraction. Neurally induced depolarizations (excitatory junction potentials) are not blocked and are sometimes augmented by the nonselective alpha-blocker phentolamine even though the depolarization induced by norepinephrine is blocked by phentolamine. Excitatory junction potentials are antagonized by the alpha 1-blocker prazosin. The contraction caused by clonidine is blocked to a greater degree by yohimbine (a relatively selective alpha 2-blocker) than by prazosin. The contraction caused by phenylephrine is blocked to a greater degree by prazosin than by yohimbine. These data indicate that phenylephrine and clonidine act by different mechanisms and, taken together with previous studies, suggest that alpha 1- and alpha 2-stimulation utilize different excitation-contraction coupling mechanisms.

scholarly journals Soluble guanylate cyclase modulators blunt hyperoxia effects on calcium responses of developing human airway smooth muscle

2015 ◽  
Vol 309 (6) ◽  
pp. L537-L542 ◽  
Author(s):  
Rodney D. Britt ◽  
Michael A. Thompson ◽  
Ine Kuipers ◽  
Alecia Stewart ◽  
Elizabeth R. Vogel ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Guanylate Cyclase ◽  
Therapeutic Strategy ◽  
Soluble Guanylate Cyclase ◽  
Novel Drugs ◽  
Intracellular Ca ◽  
Recurrent Wheeze ◽  
Airway Tone ◽  
Sgc Stimulators

Exposure to moderate hyperoxia in prematurity contributes to subsequent airway dysfunction and increases the risk of developing recurrent wheeze and asthma. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) axis modulates airway tone by regulating airway smooth muscle (ASM) intracellular Ca2+ ([Ca2+]i) and contractility. However, the effects of hyperoxia on this axis in the context of Ca2+/contractility are not known. In developing human ASM, we explored the effects of novel drugs that activate sGC independent of NO on alleviating hyperoxia (50% oxygen)-induced enhancement of Ca2+ responses to bronchoconstrictor agonists. Treatment with BAY 41–2272 (sGC stimulator) and BAY 60-2770 (sGC activator) increased cGMP levels during exposure to 50% O2. Although 50% O2 did not alter sGCα1 or sGCβ1 expression, BAY 60-2770 did increase sGCβ1 expression. BAY 41-2272 and BAY 60-2770 blunted Ca2+ responses to histamine in cells exposed to 50% O2. The effects of BAY 41-2272 and BAY 60-2770 were reversed by protein kinase G inhibition. These novel data demonstrate that BAY 41-2272 and BAY 60-2770 stimulate production of cGMP and blunt hyperoxia-induced increases in Ca2+ responses in developing ASM. Accordingly, sGC stimulators/activators may be a useful therapeutic strategy in improving bronchodilation in preterm infants.

scholarly journals Superficial buffer barrier and preferentially directed release of Ca2+ in canine airway smooth muscle

1999 ◽  
Vol 276 (5) ◽  
pp. L744-L753 ◽  
Author(s):  
Luke J. Janssen ◽  
Pierre A. Betti ◽  
Stuart J. Netherton ◽  
Denise K. Walters
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Airway Smooth Muscle ◽  
Cyclopiazonic Acid ◽  
Membrane Currents ◽  
Global Changes ◽  
Fura 2 ◽  
Mechanical Responses ◽  
Filling State

We examined cytosolic concentration of Ca2+([Ca2+]i) in canine airway smooth muscle using fura 2 fluorimetry (global changes in [Ca2+]i), membrane currents (subsarcolemmal [Ca2+]i), and contractions (deep cytosolic [Ca2+]i). Acetylcholine (10−4 M) elicited fluorimetric, electrophysiological, and mechanical responses. Caffeine (5 mM), ryanodine (0.1–30 μM), and 4-chloro-3-ethylphenol (0.1–0.3 mM), all of which trigger Ca2+-induced Ca2+ release, evoked Ca2+ transients and membrane currents but not contractions. The sarcoplasmic reticulum (SR) Ca2+-pump inhibitor cyclopiazonic acid (CPA; 10 μM) evoked Ca2+transients and contractions but not membrane currents. Caffeine occluded the response to CPA, whereas CPA occluded the response to acetylcholine. Finally, KCl contractions were augmented by CPA, ryanodine, or saturation of the SR and reduced when SR filling state was decreased before exposure to KCl. We conclude that 1) the SR forms a superficial buffer barrier dividing the cytosol into functionally distinct compartments in which [Ca2+]iis regulated independently; 2) Ca2+-induced Ca2+ release is preferentially directed toward the sarcolemma; and 3) there is no evidence for multiple, pharmacologically distinct Ca2+ pools.

scholarly journals Effect of proinflammatory cytokines on regulation of sarcoplasmic reticulum Ca2+ reuptake in human airway smooth muscle

2009 ◽  
Vol 297 (1) ◽  
pp. L26-L34 ◽  
Author(s):  
Venkatachalem Sathish ◽  
Michael A. Thompson ◽  
Jeffrey P. Bailey ◽  
Christina M. Pabelick ◽  
Y. S. Prakash ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Airway Inflammation ◽  
Airway Smooth Muscle ◽  
Proinflammatory Cytokines ◽  
Cell Types ◽  
Human Airway Smooth Muscle ◽  
Inhibitory Protein ◽  
Intracellular Ca ◽  
Additional Exposure

Airway inflammation leads to increased intracellular Ca2+ ([Ca2+]i) levels in airway smooth muscle (ASM) cells. Sarcoplasmic reticulum Ca2+ release and reuptake are key components of ASM [Ca2+]i regulation. Ca2+ reuptake occurs via sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) and is regulated by the inhibitory protein phospholamban (PLB) in many cell types. In human ASM, we tested the hypothesis that inflammation increases PLB, thus inhibiting SERCA function, and leading to maintained [Ca2+]i levels. Surprisingly, we found that human ASM does not express PLB protein (although mRNA is detectable). Overnight exposure to the proinflammatory cytokines TNFα and IL-13 did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (via CaMKII) occurs in human ASM. In fura-2-loaded human ASM cells, we found that the CaMKII antagonist KN-93 significantly slowed the rate of fall of [Ca2+]i transients induced by ACh or bradykinin (in zero extracellular Ca2+), suggesting a role for CaMKII-mediated SERCA regulation. SERCA expression was decreased by cytokine exposure, and the rate of fall of [Ca2+]i transients was slowed in cells exposed to TNFα and IL-13. Cytokine effects on Ca2+ reuptake were unaffected by additional exposure to KN-93. These data indicate that in human ASM, SERCA is regulated by mechanisms such as CaMKII and that airway inflammation maintains [Ca2+]i levels by decreasing SERCA expression and slowing Ca2+ reuptake.

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