A 20:1 synergetic mixture of cafedrine/theodrenaline accelerates particle transport velocity in murine tracheal epithelium via IP3 receptor-associated calcium release

Abstract

Background: Mucociliary clearance is a pivotal physiological mechanism that protects the lung by ridding the lower airways of pollution and colonization by pathogens, thereby preventing infections. The fixed 20:1 combination of cafedrine and theodrenaline has been used to treat perioperative hypotension or hypotensive states due to emergency situations since the 1960s. Because mucociliary clearance is impaired during mechanical ventilation and critical illness, the present study aimed to evaluate the influence of cafedrine/theodrenaline on mucociliary clearance. Material and Methods: The particle transport velocity (PTV) of murine trachea preparations was measured as a surrogate for mucociliary clearance under the influence of cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone. Inhibitory substances were applied to elucidate relevant signal transduction cascades. Results: All three applications of the combination of cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone induced a sharp increase in PTV in a concentration-dependent manner with median effective concentrations of 0.46 µM (consisting of 9.6 µM cafedrine and 0.46 µM theodrenaline), 408 and 4 μM, respectively. The signal transduction cascades were similar for the effects of both cafedrine and theodrenaline at the murine respiratory epithelium. While PTV remained at its baseline value after non-selective inhibition of β-adrenergic receptors and selective inhibition of β₁ receptors, cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone increased PTV despite the inhibition of the protein kinase A. However, IP₃ receptor activation was found to be the pivotal mechanism leading to the increase in murine PTV, which was abolished when IP₃ receptors were inhibited. Depleting intracellular calcium stores with caffeine confirmed calcium as another crucial messenger altering the PTV after the application of cafedrine/theodrenaline. Discussion: Cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone exert their effects via IP₃ receptor-associated calcium release that is ultimately triggered by β₁-adrenergic receptor stimulation. Synergistic effects at the β₁-adrenergic receptor are highly relevant to alter the PTV of the respiratory epithelium at clinically relevant concentrations. Further investigations are needed to assess the value of cafedrine/theodrenaline-mediated alterations in mucociliary function in clinical practice.

Keywords: Akrinor; ciliary activity; ciliary beat frequency; mucociliary clearance; perioperative.

FIGURE 1

Tracheal preparation, microscopy, and particle tracking. (A) The trachea is dissected with a parasternal incision of the thorax and a median incision of the throat. (B) The trachea was fixed so that the pars membranacea, including the musculus trachealis, faced upward. (C) Particle tracks were recorded and processed offline.

FIGURE 2

Particle transport velocity (PTV) is increased by cafedrine/theodrenaline, cafedrine alone and theodrenaline alone. Application of (A) 20:1 cafedrine/theodrenaline (●, 0.46 µM), (C) cafedrine alone (◼ 408 µM), and (E) theodrenaline alone (▲, 4 µM) provoked a steep and long-lasting increase in murine PTV, while the PTV remained constant around its baseline value in control experiments (○).Concentration-response relationship of (B) 20:1 cafedrine/theodrenaline, (D) cafedrine alone, and (F) theodrenaline alone, as described by the Hill equation. ⊥ standard error of the mean.

FIGURE 3

The increase in PTV depends on β-adrenergic receptor activation. Effects of (A, B) 20:1 cafedrine/theodrenaline, (C, D) cafedrine alone, and (E, F) theodrenaline alone completely vanished when β-adrenergic receptors were blocked by the non-selective inhibitor ICI. ICI alone did not provoke any alteration in the PTV. ***p < 0.001, ns: not significant. ● 20:1 cafedrine/theodrenaline, ◐ 20:1 cafedrine/theodrenaline + ICI 100 μM, ◼ cafedrine, ◧ cafedrine + ICI 100 μM, ▲ theodrenaline, ◭ theodrenaline + ICI 100 μM, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).

FIGURE 4

Alteration of PTV is completely dependent on β₁-adrenergic receptor activation. When the selective β₁-adrenergic receptor blocker CGP (100 µM) was applied, the effects of (A, B) 20:1 cafedrine/theodrenaline, (C, D) cafedrine alone, and (E, F) theodrenaline alone completely vanished. CGP alone did not provoke any alteration in the PTV. ***p < 0.001, ns: not significant. ● 20:1 cafedrine/theodrenaline, ◐ 20:1 cafedrine/theodrenaline + CGP 100 μM, ◼ cafedrine, ◧ cafedrine + CGP 100 μM, ▲ theodrenaline, ◭ theodrenaline + CGP 100 μM, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).

FIGURE 5

The increase in PTV was independent from protein kinase A (PKA). Baseline PTV decreased when the selective PKA inhibitor H-89 (10 µM) was applied. However, (A, B) 20:1 cafedrine/theodrenaline, (C, D) cafedrine alone, and (E, F) theodrenaline alone still provoked a steep increase in PTV. **p < 0.01, ***p < 0.001, ns: not significant. ● 20:1 cafedrine/theodrenaline, ◐ 20:1 cafedrine/theodrenaline + H-89 10 μM, ◼ cafedrine, ◧ cafedrine + H-89 10 μM, ▲ theodrenaline, ◭ theodrenaline + H-89 10 μM, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).

FIGURE 6

The increase in PTV was independent from phospholipase C (PLC). When the selective PLC inhibitor U-73122 (7.5 µM) was applied, (A, B) 20:1 cafedrine/theodrenaline, (C, D) cafedrine alone, and (E, F) theodrenaline alone still provoked a steep increase in PTV. ***p < 0.001, ns: not significant. ● 20:1 cafedrine/theodrenaline, ◐ 20:1 cafedrine/theodrenaline + U-73122 7.5 µM, ◼ cafedrine, ◧ cafedrine + U-73122 7.5 µM, ▲ theodrenaline, ◭ theodrenaline + U-73122 7.5 µM, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).

FIGURE 7

IP₃ receptor activation was found to be the pivotal mechanism leading to the increase in PTV. IP₃ receptors were selectively inhibited by 2-aminoethoxydiphenylborane (2-APB, 40 µM). When (A, B) 20:1 cafedrine/theodrenaline, (C, D) cafedrine alone, or (E, F) theodrenaline alone were subsequently applied, no increase in PTV was observed compared to control experiments. ***p < 0.001, ns: not significant. ● 20:1 cafedrine/theodrenaline, ◐ 20:1 cafedrine/theodrenaline + 2-APB 40 μM, ◼ cafedrine, ◧ cafedrine + 2-APB 40 μM, ▲ theodrenaline, ◭ theodrenaline + 2-APB 40 μM, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).

FIGURE 8

Intracellular Ca²⁺ stores were depleted by caffeine (30 mM), which subsequently decreased PTV. When (A) 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone were applied, a decrease from baseline was inexorable and (B) no increase was observed. ***p < 0.001, ns: not significant. ○ caffeine 30 mM, ● 20:1 cafedrine/theodrenaline + caffeine 30 mM, ◼ cafedrine + caffeine 30 mM, ▲ theodrenaline + caffeine 30 mM, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).

FIGURE 9

The PTV increase is largely independent from extracellular Ca²⁺ entry. PTV was measured in Ca²⁺-free buffer solution after (A, B) 20:1 cafedrine/theodrenaline and (E, F) theodrenaline alone were applied. However, PTV was significantly lower when PTV triggered by (C, D) cafedrine was compared to Ca²⁺-containing buffer solution. **p < 0.01, ***p < 0.001, ns: not significant. ● 20:1 cafedrine/theodrenaline, ○ 20:1 cafedrine/theodrenaline in Ca²⁺-free buffer, ◼ cafedrine, □ cafedrine in Ca²⁺-free buffer, ▲ theodrenaline, ◇ theodrenaline in Ca²⁺-free buffer, ⊥ standard error of the mean, box and whisker plots indicate median, interquartile range (box), minimum and maximum (whiskers).