Our experiments revealed that 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone increased [Ca²⁺]_i, when applied to human tracheal epithelial cells. We observed sharp, transient peaks in the FURA-2 340/380 ratio, which followed a dose–response relationship and were described by the Hill equation. Herein, it should be noted that the effect curves of 20:1 cafedrine/theodrenaline and cafedrine alone showed a profile where the increase from 0 to 100% is already achieved within one log unit, which would be classically attributed to ion channel activation. In contrast, only the dose–response curve of theodrenaline clearly indicated receptor-mediated effects because the increase from 0 to 100% required two log units. However, although the formal criteria for receptor-mediated Ca²⁺ release were only barely met in our dose–response curves following the application of 20:1 cafedrine/theodrenaline and cafedrine alone, our following experiments clearly proved adrenergic and ryanodine receptor-mediated Ca²⁺ release following the application of all three substances analyzed in our experiments. Interestingly, the EC₅₀ of cafedrine alone and theodrenaline alone were within comparable ranges; however, when applied as the clinically used 20:1 mixture, much more cafedrine was required to achieve a significant effect on [Ca²⁺]_i, and the applied concentration of theodrenaline seemed almost negligible in the light of the calculated EC₅₀ of theodrenaline alone. Moreover, [Ca²⁺]_i peaks observed after the application of 20:1 cafedrine/theodrenaline were consistently lower than those observed following applications of the individual substances alone. Interestingly, clinically irrelevant high concentrations of 20:1 cafedrine/theodrenaline ultimately led to cell lysis, which was also observed following the administration of high concentrations of cafedrine or theodrenaline alone; however we never observed a higher FURA-2 340/380 ratio than that shown in our dose–response curves. Although the pharmacokinetics of cafedrine and theodrenaline remain largely unknown, the immediate response in blood pressure was attributed to theodrenaline, while the effects of cafedrine were observed after a 20-min delay^17,20. Therefore, it has been hypothesized that cafedrine may not provoke any sympathomimetic actions alone; it may be metabolized into active metabolites that exert its clinical effects¹⁷. In our experiments, the effect observed after the application of 20:1 cafedrine/theodrenaline could only be attributed to theodrenaline alone if strong synergistic effects would apply in the presence of cafedrine. Because the concentration of theodrenaline alone in the 20:1 mixture was insufficient to provoke any significant changes in [Ca²⁺]_i, we conclude that the foremost effect on changing [Ca²⁺]_i in our experiments was induced by cafedrine. This assumption is supported by the observation that very high concentrations of cafedrine were applied using the 20:1 combination compared with the dose–response relationship of cafedrine alone. However, our model cannot represent the effects of potential drug metabolites, as we used isolated cells, and the drugs were directly applied to the buffer solution. Furthermore, clinical data reported a plasma concentration of 6 µg/ml after the intravenous application of one ampoule of 2 ml cafedrine/theodrenaline; therefore, all concentrations used in our experiments were significantly higher than clinically used concentrations²⁰. However, the transient intravascular and intraepithelial concentrations immediately after injection are unknown and might be much higher than those observed after distribution to the body compartments; therefore, our data should be interpreted with caution, and further studies are necessary to evaluate the transferability of the concentrations used in our experiments.

After the transient peak, during which Ca²⁺ directly increases ciliary beat frequency and calmodulin-bound Ca²⁺ activated cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP)-dependent pathways, [Ca²⁺]_i rapidly returned to its baseline³. The rapid restoration of baseline [Ca²⁺]_i is primarily explained by SERCA activity, which pumps Ca²⁺ into the ER after cytosolic Ca²⁺ release, or by mitochondrial Ca²⁺ buffering^21,22. However, almost every other small cell organelle contributes to the rapid restoration of baseline [Ca²⁺]_i, and prolonged alteration of ciliary beat frequency is induced by transient changes in [Ca²⁺]_i. Immediately after purinergic or cholinergic stimulation, tracheal epithelial cells exhibited a transient increase in [Ca²⁺]_i; however, ciliary beat frequency remained consistently increased^23,24. Therefore, we conclude that analogous mechanisms are mediated after the stimulation of β-adrenergic receptors, which were the pivotal receptors involved in [Ca²⁺]_i alterations in our experiments. The increase in [Ca²⁺]_i completely vanished after 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone were added during non-selective adrenergic receptor inhibition. These results are in line with the clinical effect of cafedrine/theodrenaline, which increases cardiac stroke volume via β₁-adrenergic receptor activation¹⁴. In-vitro studies using human atrial myocardium and coronary arteries also elucidated β₁-adrenergic receptor activation as a pivotal mechanism; however, effects on α-adrenergic receptors were observed in arteries after β-adrenergic receptor inhibition^17,25. Although these effects were attributed to theodrenaline alone, we did not observe similar effects in human tracheal epithelial cells. These observations are in line with the data reported by Weiterer et al., who reported the exclusive presence of the α1D-adrenergic receptor subtype in murine tracheal epithelium²⁶. However, murine particle transport velocity was independent from α-adrenergic receptor activation. Therefore, we conclude that α-receptor activation might occur in human tracheal epithelial cells following the administration of cafedrine/theodrenaline, but no influence on [Ca²⁺]_i or mucociliary clearance could be detected in light of the available in-vitro data²⁶. The theophylline component of cafedrine and theodrenaline is believed to inhibit PDE, which should lead to the persistence of second messengers, such as cGMP and cAMP, improving cardiac inotropy¹⁴. However, only high, clinically irrelevant concentrations of cafedrine/theodrenaline were able to provoke significant inhibition of PDE in human atrial myocardium¹⁷. Although we used high concentrations of cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone, we were only able to detect β-adrenergic receptor stimulation because the increase in [Ca²⁺]_i completely vanished in the presence of β-adrenergic receptor inhibition. When other signal transduction cascades were involved, we should have detected a persistent increase in [Ca²⁺]_i. This finding is underlined by the knowledge that PDE inhibition might not influence ciliary beat frequency or mucociliary clearance to a clinically relevant degree, because data remain controversial regarding the alteration of mucociliary clearance following treatment with theophylline^27,28,29.

When we used Ca²⁺-free buffer solution, [Ca²⁺]_i increased to a significantly lesser degree following administration of cafedrine alone and theodrenaline alone than the increase observed in Ca²⁺-containing buffer solution. Therefore, extracellular Ca²⁺ influx contributes to the rise in [Ca²⁺]_i, which is foremost realized through SOCE in non-excitable cells following Ca²⁺ release from internal stores^12,13. ORAI proteins, which are mediated by stromal interaction molecule proteins, are the most important mediators of SOCE¹². Therefore, further experiments elucidating receptor expression and distinct signal transduction pathways should be performed, although their specific inhibition is complicated due to their diverse interactions and multiple targets located on the plasma membrane¹³. However, because the [Ca²⁺]_i peak in the Ca²⁺-free buffer solution was comparable to the peak observed in Ca²⁺-containing buffer solution following the administration of 20:1 cafedrine/theodrenaline, the clinical relevance of SOCE following the administration of cafedrine/theodrenaline in human tracheal epithelial cells remains questionable. Internal stores were depleted to detect the intracellular stores, which released Ca²⁺ following the administration of cafedrine/theodrenaline. When mitochondrial Ca²⁺ stores were depleted, [Ca²⁺]_i continued to increase following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone. Therefore, we conclude that cafedrine/theodrenaline does not depolarize mitochondrial membrane potential, which would lead to Ca²⁺ release from these stores. However, [Ca²⁺]_i peaks following the administration of 20:1 cafedrine/theodrenaline and cafedrine alone were significantly higher than those observed without prior mitochondrial store depletion. This observation confirms the mitochondrial ability to buffer Ca²⁺ ions via the mitochondrial Ca²⁺ uniporter when [Ca²⁺]_i exceeds 500 nM³⁰. Interestingly, [Ca²⁺]_i remained higher for the rest of the observation time. Because DNP administration decouples oxidative phosphorylation without altering cytosolic pH, less ATP supplying the ATP-dependent SERCA might be available in these experiments, leading to the observation of persistent [Ca²⁺]_i baseline shift. However, SERCA was still able to handle the transient peak [Ca²⁺]_i by altering [Ca²⁺]_i close to its former baseline value. This finding underlines our conclusion that the elevated peak in [Ca²⁺]_i is foremost triggered by the inhibited mitochondrial Ca²⁺ capacity and not by a lack of ATP, which might impair SERCA activity.

Because the increase in [Ca²⁺]_i completely vanished following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone, when RyR were inhibited, we conclude that cafedrine/theodrenaline releases Ca²⁺ from the ER exclusively by RyR activation. In general, Ca²⁺ release from the ER is achieved via IP₃ receptor or RyR activation³¹. However, our experiments revealed equal peaks of [Ca²⁺]_i when IP₃ receptors were inhibited, and the increase in [Ca²⁺]_i completely vanished after RyR inhibition. Therefore, we conclude that Ca²⁺ release following β-adrenergic receptor activation depends solely on RyR activation, and IP₃ receptor-associated Ca²⁺ release does not occur in human tracheal epithelial cells following cafedrine/theodrenaline administration.

Consequently, RyR-2 and RyR-3 expression was revealed using RT-PCR in murine tracheal epithelium. While RyR-3 is co-expressed with RyR-1 or RyR-2 in many tissues, RyR 2 has mainly been studied in cardiac muscle cells; however, RyR-2 expression has been demonstrated in smooth muscle cells and non-excitable cells, such as pancreatic acinar cells and kidney epithelial cells^32,33,34,35. Therefore, RyR-2 expression in the tracheal epithelium is in line with its expression in other non-excitable cells. While RyR activation is achieved following various signal transduction cascades, the best-known mechanism is the Ca²⁺-induced Ca²⁺-release, whereby RyR-2 activation is triggered by a local increase of [Ca²⁺]_i³⁶. Local increase in [Ca²⁺]_i can be realized through nearby RyR activation, or IP₃ receptor activation; however, our data indicate that RyR activation was independent from IP₃ receptor activation following cafedrine/theodrenaline administration³⁷. Therefore, alternative RyR activation following β-adrenergic receptor stimulation must be considered in tracheal epithelial cells. β-adrenergic stimulation has been shown to increase RyR-2 activity in cardiac muscle cells via intracellular-mediated Ca²⁺ and Mg²⁺ regulation, and receptor phosphorylation³⁸. Furthermore, protein kinase A and cAMP, which are both pivotal messengers following the β₁-signal transduction cascade, have been shown to induce Ca²⁺ release via RyR-2 in cardiac muscles and non-excitable cells, respectively^39,40. In addition, adrenergic receptor signaling increases nicotinic acid adenine dinucleotide phosphate and cyclic adenosine diphosphate-ribose levels, which both activate RyR-2-associated Ca²⁺ release^33,41. Because RyR-3 is more readily activated by an increase in local [Ca²⁺]_i compared with RyR-2, its activation in tracheal epithelial cells following the administration of cafedrine/theodrenaline can be achieved following RyR 2-associated Ca²⁺ efflux⁴². However, as discrepancies in receptor expression between mammals could not be excluded, further studies in human tissues should be conducted to confirm RyR expression. Local distribution, and the RyR-2-to-RyR-3 ratio in tracheal epithelial cells could be evaluated using immunohistochemistry.

Several limitations of our experiments must be acknowledged. First, we used isolated tracheal epithelial cells; therefore, the physiological integrity of a respiratory tract was not preserved, including a lack of basal tissues and cell–cell junctions. Therefore, physiological drug administration via capillary vessels could not be replicated, and atypical entrance (e.g. from the apical or lateral side of the cells) of our tested drugs was preserved. Second, we used high concentrations of cafedrine/theodrenaline, cafedrine, and theodrenaline to achieve alterations in [Ca²⁺]_i and clinically administered concentrations are much lower; therefore, it is possible that we observed [Ca²⁺]_i kinetics that do not occur when lower concentrations are used in vivo. Third, some concentrations applied to achieve the specific dose–response curves were higher than was warranted by the integrity of the observed cells; therefore, a maximum FURA-2 340/380 ratio following substance administration was set at the last valid observed value. However, concentrations applied to assess the distinct signal transduction cascades did prove the validity of the calculated dose–response curves in our experimental setting. Fourth, our measurement of [Ca²⁺]_i using the FURA-2 340/380 ratio was not calibrated; therefore, we can only report relative alterations in [Ca²⁺]_i, and no absolute concentrations were measured.

In conclusion, we provide evidence that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone induce the release of Ca²⁺ from caffeine-sensitive internal stores that is exclusively triggered by β-adrenergic receptor stimulation, resulting in RyR activation. RT-PCR revealed the presence of RyR-2 and RyR-3 in mammalian cells, and the relevant influence of extracellular Ca²⁺ influx was only observed after the application of cafedrine alone or theodrenaline alone. However, clinical plasma concentrations are considerably lower than those used in our experiments to trigger a significant increase in [Ca²⁺]_i; therefore, further studies are needed to alter the ability of cafedrine/theodrenaline to change mucociliary clearance in clinical practice.