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The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles.

Perez JF, Sanderson MJ - J. Gen. Physiol. (2005)

Bottom Line: High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs.By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching.Caffeine abolished the contractile effects of 5-HT, ACH, and KCl.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations.

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Effect of caffeine and CPA on airway contraction induced by KCl. (A) Airways were sequentially stimulated with 50 mM KCl in the absence or presence of 20 mM caffeine (top bars). (B) A line-scan obtained from phase-contrast images in a region similar to that shown by the dotted line in Fig. 5 A during sequential stimulation with KCl in the absence or presence of 10 μM CPA (top bars). Twitches induced by KCl are observed as transient signals toward the lumen of the airway and represent the local displacement of cells produced by the contraction of one or a few SMCs. A few spontaneous twitches were observed before exposure to KCl. Representative data of at least four experiments from different slices from three mice.
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fig12: Effect of caffeine and CPA on airway contraction induced by KCl. (A) Airways were sequentially stimulated with 50 mM KCl in the absence or presence of 20 mM caffeine (top bars). (B) A line-scan obtained from phase-contrast images in a region similar to that shown by the dotted line in Fig. 5 A during sequential stimulation with KCl in the absence or presence of 10 μM CPA (top bars). Twitches induced by KCl are observed as transient signals toward the lumen of the airway and represent the local displacement of cells produced by the contraction of one or a few SMCs. A few spontaneous twitches were observed before exposure to KCl. Representative data of at least four experiments from different slices from three mice.

Mentions: Although we believe that KCl is acting via membrane depolarization of the SMCs, it is possible that KCl may also act via membrane depolarization of neural terminals within the lung slice to locally release neurotransmitters. However, we found that KCl induced contraction of the airways in the presence of atropine and ketanserin, receptor antagonists that fully blocked the effect of ACH and 5-HT, respectively. Similarly, the possibility that KCl acts via the release of an α1-agonist was ruled out by the fact that the airways were totally unresponsive to phenylephrine. Because ATP (but not AMP or adenosine) can stimulate airway contraction (Bergner and Sanderson, 2002b) and may be coreleased from nerve terminals, we added 10 U/ml apyrase, a potent ATPase, during KCl stimulation. Under these conditions, a twitching response of the airways similar to that induced by KCl alone was observed. In addition, it should be noted that during agonist or KCl exposure, the lung slices were under constant perfusion, a condition that would be expected to quickly wash away any small concentrations of agonists released from nerves. However, the twitching response induced by KCl persisted with an approximately constant frequency during several minutes of perfusion with KCl. Furthermore, this response was elicited by several sequential stimulations with KCl (Fig. 2 B, Fig. 5 B, and Fig. 12), which would have been expected to run-down the release of agonists from isolated nerve terminals.


The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles.

Perez JF, Sanderson MJ - J. Gen. Physiol. (2005)

Effect of caffeine and CPA on airway contraction induced by KCl. (A) Airways were sequentially stimulated with 50 mM KCl in the absence or presence of 20 mM caffeine (top bars). (B) A line-scan obtained from phase-contrast images in a region similar to that shown by the dotted line in Fig. 5 A during sequential stimulation with KCl in the absence or presence of 10 μM CPA (top bars). Twitches induced by KCl are observed as transient signals toward the lumen of the airway and represent the local displacement of cells produced by the contraction of one or a few SMCs. A few spontaneous twitches were observed before exposure to KCl. Representative data of at least four experiments from different slices from three mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234076&req=5

fig12: Effect of caffeine and CPA on airway contraction induced by KCl. (A) Airways were sequentially stimulated with 50 mM KCl in the absence or presence of 20 mM caffeine (top bars). (B) A line-scan obtained from phase-contrast images in a region similar to that shown by the dotted line in Fig. 5 A during sequential stimulation with KCl in the absence or presence of 10 μM CPA (top bars). Twitches induced by KCl are observed as transient signals toward the lumen of the airway and represent the local displacement of cells produced by the contraction of one or a few SMCs. A few spontaneous twitches were observed before exposure to KCl. Representative data of at least four experiments from different slices from three mice.
Mentions: Although we believe that KCl is acting via membrane depolarization of the SMCs, it is possible that KCl may also act via membrane depolarization of neural terminals within the lung slice to locally release neurotransmitters. However, we found that KCl induced contraction of the airways in the presence of atropine and ketanserin, receptor antagonists that fully blocked the effect of ACH and 5-HT, respectively. Similarly, the possibility that KCl acts via the release of an α1-agonist was ruled out by the fact that the airways were totally unresponsive to phenylephrine. Because ATP (but not AMP or adenosine) can stimulate airway contraction (Bergner and Sanderson, 2002b) and may be coreleased from nerve terminals, we added 10 U/ml apyrase, a potent ATPase, during KCl stimulation. Under these conditions, a twitching response of the airways similar to that induced by KCl alone was observed. In addition, it should be noted that during agonist or KCl exposure, the lung slices were under constant perfusion, a condition that would be expected to quickly wash away any small concentrations of agonists released from nerves. However, the twitching response induced by KCl persisted with an approximately constant frequency during several minutes of perfusion with KCl. Furthermore, this response was elicited by several sequential stimulations with KCl (Fig. 2 B, Fig. 5 B, and Fig. 12), which would have been expected to run-down the release of agonists from isolated nerve terminals.

Bottom Line: High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs.By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching.Caffeine abolished the contractile effects of 5-HT, ACH, and KCl.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations.

Show MeSH
Related in: MedlinePlus