Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on “Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells”

Background The authors investigated the effects of intravenous anesthetics on alpha-adrenergic-induced oscillations in intracellular free calcium concentration ([Ca2+]i) in individual pulmonary artery smooth muscle cells (PASMCs). Methods PASMCs were cultured from explants of canine intrapulmonary artery. Fura-2-loaded PASMCs were continuously superfused with phenylephrine (10 microM) at 37 degrees C on the stage of an inverted fluorescence microscope. Measurement of [Ca2+]i was via a dual wavelength spectrofluorometer. Intravenous anesthetics were added to the superfusate to assess their effects on the phenylephrine-induced [Ca2+]i oscillations. Results Resting [Ca2+]i was 103 +/- 6 nM. Phenylephrine stimulated [Ca2+]i oscillations, reaching a peak concentration of 632 +/- 20 nM and a frequency of 1.53 +/- 0.14 transients/min. The effects of phenylephrine were dose-dependent. The effects of intravenous anesthetics on phenylephrine-induced [Ca2+]i oscillations were dose-dependent. Ketamine (100 microM) reduced the amplitude (221 +/- 22 nM) but not the frequency (1.48 +/- 0.11/min) of the oscillations, whereas thiopental (100 microM) decreased the amplitude (270 +/- 20 nM) and the frequency (1.04 +/- 0.10/min). Propofol (100 microM) and the Intralipid vehicle inhibited the amplitude (274 +/- 11 nM) but not the frequency (1.39 +/- 0.11/min) of the oscillations. The effects of ketamine and thiopental, but not propofol, were evident at clinically relevant concentrations. Conclusion Ketamine, thiopental, and propofol exerted differential effects to inhibit the amplitude or the frequency of phenylephrine-induced [Ca2+]i oscillations in individual PASMCs. Thus, intravenous anesthetics may alter the pulmonary vascular response to alpha-adrenoreceptor activation by directly inhibiting [Ca2+]i signaling in PASMCs.

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Benzodiazepines Differentially Inhibit Phenylephrine-induced Calcium Oscillations in Pulmonary Artery Smooth Muscle Cells

Anesthesiology ◽

10.1097/00000542-199803000-00032 ◽

1998 ◽

Vol 88 (3) ◽

pp. 792-799 ◽

Cited By ~ 26

Author(s):

Sung Jin Hong ◽

Derek S. Damron ◽

Paul A. Murray

Keyword(s):

Smooth Muscle ◽

Pulmonary Artery ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Calcium Oscillations ◽

Intracellular Free Calcium ◽

Free Calcium ◽

Critical Determinant ◽

Pulmonary Artery Smooth Muscle ◽

Dose Dependent

Background Modulation of intracellular free calcium is a critical determinant of vasomotor tone. The authors investigated the effects of three benzodiazepines on alpha-adrenergic-induced oscillations in intracellular free calcium in individual pulmonary artery smooth muscle cells. Methods Pulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura-2-loaded pulmonary artery smooth muscle cells were continuously superfused with phenylephrine (10 microM) at 37 degrees C on the stage of an inverted fluorescence microscope. Intracellular free calcium was measured using a dual wavelength spectrofluorometer. After establishment of steady-state intracellular free calcium oscillations induced by phenylephrine, lorazepam, diazepam, or midazolam was added to the superfusate. The amplitude and frequency of the intracellular free calcium oscillations were compared before and after addition of each agent. Results Resting mean +/- SEM values of intracellular free calcium were 68 +/- 8 nM. Phenylephrine stimulated dose-dependent oscillations in intracellular free calcium, which reached a peak concentration of 676 +/- 35 nM and a frequency of 1.08 +/- 0.1 transients/min. Addition of lorazepam (1 microM) inhibited (P < 0.05) the amplitude (591 +/- 32 nM) but not the frequency (0.97 +/- 0.1 transients/min) of the oscillations. Conversely, diazepam (1 microM) decreased (P < 0.05) the frequency (0.79 +/- 0.1 transients/min) but not the amplitude (663 +/- 37 nM) of the oscillations. These effects were dose-dependent. In contrast, midazolam (1-30 microM) had no effect on the amplitude or frequency of intracellular free calcium oscillations. At concentrations higher than 100 microM, however, all three benzodiazepines inhibited both the amplitude and frequency of the intracellular free calcium oscillations. Conclusion Lorazepam and diazepam but not midazolam exerted differential inhibitory effects on phenylephrine-induced intracellular free calcium oscillations. Benzodiazepines may alter the pulmonary vascular response to sympathetic alpha-adrenoreceptor activation by direct inhibition of intracellular free calcium signaling in pulmonary artery smooth muscle cells.

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