The unique ability of the respiratory tract to protect the integrity of the airways by removing potentially harmful substances is called mucociliary clearance. Mucociliary clearance protects the lungs by ridding the airways of pollution and preventing pathogen colonization, and infection. The cooperation of different cells, such as basal, suprabasal, goblet, and multiciliated epithelial cells, ensures the complex mucociliary clearance function of the respiratory tract (Lee and Foskett, 2014; Legendre et al., 2021). These ciliated epithelial cells use outward-directed transportation to prevent the accumulation of debris and colonization by microbial pathogens (Whitsett, 2018; Legendre et al., 2021). Cilia are located on the apical side of the respiratory epithelium along the airways, and are constructed from unique structural proteins (Whitsett, 2018). Ciliary activity can be quantified as ciliary beat frequency (CBF), with particle transport velocity (PTV) used as its surrogate (Delmotte and Sanderson, 2006; Weiterer et al., 2014).

Mucociliary clearance is a complex process that can adapt to different physiological and pathophysiological conditions, with many endogenous and exogenous pathways involved in modifying its function (Delmotte and Sanderson, 2006; Legendre et al., 2021). Therefore, not only inhaled particles but also mucus, electrolytes, and endogenous defensive substances can be removed from the lower airways, which are then expectorated under normal physiological conditions (Delmotte and Sanderson, 2006; Legendre et al., 2021).

The sympathetic and parasympathetic nervous systems increase or decrease ciliary activity, facilitated by their transmitters, norepinephrine and acetylcholine (Frohock et al., 2002; Hollenhorst and Krasteva-Christ, 2021). Important secondary messengers are cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate, and calcium ions (Ca²⁺), which ultimately induce alterations in the CBF (Salathe, 2007; Wyatt, 2015). However, ciliary activity is also sensitive to local temperature, acid-base balance, humidity, mechanical stress, cytokines released during infection, and paracrine effects mediated by adjacent cells, such as mast cells or macrophages (Sanderson and Dirksen, 1986; Kilgour et al., 2004; Sutto et al., 2004; Salathe, 2007; Weiterer et al., 2014; Perniss et al., 2020). Local milieu changes in pathophysiological conditions also influence mucociliary clearance, such as the formation of reactive oxygen species, and pathogenic bacterial, viral or fungal components mediate opposing effects and can reduce CBF with subsequent clinically relevant impairment in mucociliary clearance (Burman and Martin, 1986; Weiterer et al., 2015; Kamiya et al., 2020).

Some severe congenital diseases, such as primary cilia dyskinesia, are characterized by recurrent infections caused by a significant impairment of mucociliary clearance (Ren et al., 2023). However, acquired ciliary dysfunction is pivotal in many pathophysiologic conditions. For example, mucociliary clearance is impaired in tracheal intubated and mechanically ventilated patients, and mechanical ventilation is associated with increased bacterial colonization and pneumonia (Bassi et al., 2008). Furthermore, an overall impairment of mucociliary clearance was also found in critically ill patients in intensive care units (Nakagawa et al., 2005). Impairment of mucociliary clearance is already observed during general anesthesia with mechanical ventilation following endotracheal intubation and may even persist in the postoperative period (Adams et al., 2021). The temperature of the inspired air and the respirator settings modulate the degree of impairment, but the drugs used perioperatively also influence mucociliary clearance (Deng et al., 2023).

Some intravenous and volatile anesthetics, and perioperative antagonists, such as neostigmine and sugammadex, have been evaluated for their influence on mucociliary clearance (Forbes and Gamsu, 1979; Kesimci et al., 2008; Ozciftci et al., 2022). In addition, a recent experimental series showed that a drug commonly used to treat perioperative hypotension, a 20:1 mixture of cafedrine/theodrenaline, accelerated murine PTV and is, therefore, suggested to modulate mucociliary clearance when administered intravenously (Schmidt et al., 2023a). However, cafedrine/theodrenaline is solely used as an intravenous bolus applied for a short period to treat acute hypotensive states. When profound and prolonged hypotension persists, vasopressors, such as dopamine, norepinephrine, or vasopressin, are administered for longer periods in the perioperative setting and intensive care units (Ruiqiang et al., 2021; Douglas et al., 2023).

Dopamine, a precursor of norepinephrine that lacks one hydroxyl group, has been widely used to maintain adequate mean arterial pressures in different forms of shocks (Suzuki et al., 2022). Dopamine has different effects at different dosages. While there is a large inter-individual variability in the effects observed, low dosages induce vasodilation on visceral and renal vasculature via D₁ receptors, and intermediate and high dosages act on β₁- and α₁-adrenergic receptors, leading to positive inotropy and vasoconstriction, respectively (Jozwiak, 2022; Ratnani et al., 2023). Patients who received dopamine after non-cardiac surgery had comparable mortality rates to those who received norepinephrine (Aoki et al., 2023). However, dopamine was associated with longer intensive care unit stays, and it led to a higher rate of cardiac arrhythmias and mortality in patients with septic shock than norepinephrine (Avni et al., 2015; Ruiqiang et al., 2021; Aoki et al., 2023). Therefore, norepinephrine has emerged as the first-line vasopressor recommended for patients with septic shock (Ruiqiang et al., 2021).

Norepinephrine is an endogenous catecholamine that is not only used in patients with septic shock but also in the perioperative setting when mean arterial pressure remains unstable after bolus injections, such as phenylephrine, ephedrine, or 20:1 cafedrine/theodrenaline (Kim et al., 2023). Norepinephrine primarily acts as an agonist of β₁-and α₁-adrenergic receptors, leading to increased inotropy and vasoconstriction, while the heart rate is mostly unaffected (Ruiqiang et al., 2021). Norepinephrine is currently the most frequently administered vasopressor to treat hypotensive states in various settings. Nevertheless, in patients with septic shock, adding vasopressin is suggested when inadequate mean arterial pressures persist despite higher norepinephrine doses (Ruiqiang et al., 2021).

Vasopressin is normally synthesized in the pituitary gland and acts on vasopressin receptors as an endogenous peptide hormone (Ruiqiang et al., 2021). While V₁ receptors lead to vasoconstriction, renal V₂ receptors have antidiuretic effects (Ammar et al., 2022). Vasopressin is used as a potent vasopressor in septic shock along with norepinephrine and other vasopressors to reduce the overall catecholamine dosages (Ruiqiang et al., 2021; Ammar et al., 2022). Furthermore, it is suggested to have beneficial effects on hypotensive states in patients with elevated pulmonary vascular resistance because it increases systemic vascular resistance but not pulmonary vascular resistance (Sarkar et al., 2015).

Since the impact of these three commonly used vasopressors on mucociliary clearance has not yet been investigated in detail, our experiments aimed to evaluate their influence on the PTV, which indicates mucociliary clearance in the lower airways, and reveal the specific signaling cascades through which they alter the PTV in the epithelium. Dopamine, norepinephrine, or vasopressin were applied to mouse tracheae, and the PTV was measured. Reverse transcription polymerase chain reactions (RT-PCR) were performed to assess the expression of vasopressin receptors in the murine trachea and isolated tracheal epithelium. Further experiments were conducted to determine the importance of calcium release, its origin, and the value of extracellular calcium entry.

Our study revealed that dopamine, norepinephrine, and vasopressin accelerate the murine PTV, suggesting changes in mucociliary clearance of the respiratory epithelium. All three analyzed substances showed concentration-response relationships, which can be described with the Hill equation, indicating specific receptor-mediated effects. Each substance ultimately leads to intracellular Ca²⁺ release, mediating the increase in the PTV, intensified by additional extracellular Ca²⁺ entry. However, dopamine, norepinephrine, and vasopressin use different, substance specific membrane-bound receptors to induce these effects.

Our experiments revealed that dopamine increases the murine PTV via the D₁/D₅ receptors, which are frequently referred to D₁-like receptors (Montalant et al., 2023). These G protein-coupled receptors induce the formation of cAMP, which ultimately activates the PKA (Bozzi and Borrelli, 2013). Our experiments suggested that PKA is the pivotal enzyme in the signal transduction cascade that increases the PTV after dopamine administration. Therefore, our results are consistent with the known signal transduction cascade after D₁-like receptor activation (Montalant et al., 2023). Ca²⁺ release was ultimately triggered by IP₃ receptor activation, which was shown by the complete inhibition of any increase in the PTV after IP₃ receptor inhibition. IP₃ receptors are generally regulated by both cAMP- and PKA-dependent pathways (Taylor, 2017). PKA phosphorylates IP₃ receptors after increasing cAMP concentrations, causing rapid, transient, and reversible changes in IP₃ receptor channel function (Betzenhauser and Yule, 2010). While phosphorylation does not directly open IP₃ receptors, phosphorylated IP₃ receptors showed an increased open probability when activated by IP₃, and closed receptor states were destabilized (Taylor, 2017). Therefore, PKA-induced phosphorylation can approximately double IP₃ receptor-mediated Ca²⁺ release (Nakade et al., 1994). While closely related, each subtype has different phosphorylation sites, resulting in only the activity of subtypes 1 and 2 but not subtype 3 being enhanced by PKA-induced phosphorylation (Nakade et al., 1994; Taylor, 2017). Moreover, cAMP has also been shown to directly affect IP₃ receptors without the concomitant activation of PKA (Taylor, 2017). However, these effects were primarily described in combination with parathyroid hormone, and it remains unclear whether they might also apply to other substances and especially to respiratory tissues (Tovey et al., 2008; Tovey et al., 2010).

Our experiments also suggested that dopamine affects β-adrenergic receptors to a lesser extent, proven by the PKA not being the pivotal receptor enzyme with norepinephrine in our experiments, and other β-adrenergic agonists in recent studies (Schmidt et al., 2023a). However, it must be noted that signal transduction after dopamine administration was only examined with one concentration reflecting the calculated EC₅₀. Because dopamine generally showed concentration-response kinetics according to the Hill equation, it appears that D₁-like receptors remain the pivotal receptor triggering increase in the PTV, with other adrenergic receptors barely affected. Therefore, only β-adrenergic receptors appear relevant, because no PTV-altering effect was found after α-receptor activation, and RT-PCR only detected the expression of the α₁D subunit in murine respiratory epithelium in other studies (Weiterer et al., 2015).

Norepinephrine induced a steeper increase in the PTV than dopamine and vasopressin. Its observed kinetics were comparable with other β-adrenergic receptor agonists, such as theodrenaline, which has been recently examined (Schmidt et al., 2023b). We observed that norepinephrine alters the PTV via unselective β-adrenergic receptor stimulation because the PTV was further reduced when the concentration of CGP20712A was increased from selective β₁-adrenergic inhibition to unselective β-adrenergic receptor inhibition. However, even the high concentration could not entirely prevent the increase in the PTV, although PTV was considerably reduced.

Therefore, we conclude that norepinephrine might increase the PTV via unselective β-adrenergic receptor activation since it is already known that PTV increases are independent of α-adrenergic receptor activation (Weiterer et al., 2015). Furthermore, our experiments showed independence from PLC, strengthening this finding because PLC is the main effector enzyme of the α₁-adrenergic receptor. Nevertheless, since the slope of the concentration-response curve for norepinephrine was very steep, it seems possible that the calculated EC₅₀ represents a slightly right-shifted position on the actual concentration-response curve. Therefore, the inhibitory concentration used in our experimental setup may not be sufficient to block a potentially higher norepinephrine concentration. Interestingly, PKA was not the pivotal signal transduction enzyme in the increase in the PTV after β-adrenergic receptor stimulation. This finding has also been observed after applying cafedrine and theodrenaline, which are both regarded as selective β₁ receptor agonists (Schmidt et al., 2023b). Notably, the effects of these β₁ receptor agonists were ultimately mediated via IP₃ receptors which was not observed in our experiments with norepinephrine. Therefore, IP₃ receptors might contribute to Ca²⁺ release because the maximum PTV was slower with than without IP₃ receptor inhibition. Different signal transduction mechanisms and receptor involvement could also be hypothesized. The linkage between β-adrenergic receptors and ryanodine receptor activation releasing Ca²⁺ from the ER or sarcoplasmic reticulum in cardiomyocytes is generally well known (Zhou et al., 2009; Berisha et al., 2021). Ryanodine receptors 2 and 3 were found in the murine respiratory epithelium (Schmidt et al., 2023b). However, allosteric modulation of ryanodine receptors by cAMP, which could lead to Ca²⁺ release and the subsequent increase in the PTV despite its independence from PKA, has only been described for ryanodine receptor 1 (Cholak et al., 2023). Therefore, further studies should be performed with other β-agonists, which might confirm our findings, and inhibitory substances, which could elucidate the distinct signal transduction cascades.

Our experiments suggested that vasopressin accelerated the murine PTV via IP₃ receptor triggered Ca²⁺ release from the ER. The relevance of extracellular Ca²⁺ entry after vasopressin administration was underscored by the markedly reduced PTV in the experiments using a Ca²⁺-free buffer solution. While the experiments with H-89 showed that PKA does not influence the change in PTV after vasopressin administration, the experiments with U-73122 suggested that PLC significantly influenced the change. These results are consistent with the typical V₁ receptor signal transduction cascade because these G-coupled receptors activate PLC and ultimately release Ca²⁺ from the ER via IP₃ receptor activation (Birnbaumer, 2000). In contrast, PKA is the main effector enzyme of V₂ receptors, whose relevance would have been suggested if the PTV had been modulated by PKA inhibition (Birnbaumer, 2000). Our experiment with the nonspecific V_1a and V₂ receptor inhibitor conivaptan initially suggested the relevance of one of these receptors. However, the increase in the PTV entirely disappearing when the V_1a receptors were selectively inhibited precluded further experiments with selective V₂ inhibitors. Furthermore, V_1b receptor inhibition did not prevent the increase in the PTV after vasopressin administration, and subsequent RT-PCR only detected the expression of V_1a in the murine trachea and tracheal epithelium. Further experiments including immunohistochemistry could focus on the detailed characterization and localization of vasopressin receptors in the murine trachea and tracheal epithelium. While the presence of vasopressin receptors in mammalian lung tissues is generally accepted, their physiological function has not been widely examined (Tahara et al., 1998; Bernard et al., 2005). Vasopressin has been shown to regulate chloride secretion in human bronchial epithelial cells, suggesting that it can complexly regulate mucociliary clearance function not only by increasing the active outward transportation processes but also mucus formation (Bernard et al., 2005). Consistent with our results, V₁ receptors also stimulated mucus production. Furthermore, even V₂ receptors were highly relevant depending on the chloride concentration gradients between the mucosal and serosal sides of the epithelial cells (Bernard et al., 2005). Tamaoki et al. found that vasopressin increased the CBF of rabbit tracheal epithelium via Ca²⁺ release by thapsiargin-sensitive stores (Tamaoki et al., 1998). Since this study only measured CBF, its authors speculated that the observed effect of vasopressin might be translated into enhanced mucociliary transport, which our experiments have now confirmed (Tamaoki et al., 1998).

Our experiments have increased the knowledge about the roles of PLC and IP₃ receptors in releasing intracellular Ca²⁺ after vasopressin receptor stimulation in murine tracheal tissues. Interestingly, some of our findings show important differences from those of Tamaoki et al. and must be discussed (Tamaoki et al., 1998). Firstly, Tamaoki et al. found no significant difference in the rabbit CBF increase when extracellular Ca²⁺ was depleted. In contrast, our experiments showed an impressive reduction in the murine PTV following the application of vasopressin in a Ca²⁺-free buffer solution. Secondly, while the relevance of the V₁ receptor pathway is unquestionable in both studies, their results differ regarding whether the V_1a or V_1b receptor is the main receptor responsible for the observed effects.

When the chloride secretion ability of vasopressin receptors was examined in human bronchial epithelial cell lines, vasopressin’s effects were completely inhibited when both receptor subtypes were separately inhibited by highly specific inhibitors, suggesting the involvement of a V₁-like receptor (Bernard et al., 2005). However, both V_1a and V_1b receptors were detected in subsequent RT-PCR (Bernard et al., 2005). Consistent with our experiments, Tamaoki et al. demonstrated V₁ receptor involvement in the rabbit CBF by its increase disappearing after applying OPC-21268, an unselective V₁ receptor inhibitor (Tamaoki et al., 1998). However, when they measured intracellular Ca²⁺ concentrations after administering selective V_1a or V_1b agonists, they increased more with the V_1b agonist and were not significantly reduced by the selective V_1a inhibitor CTM-vasopressin (Bernard et al., 2005). Whether these inconsistent results reflect differences between mammalians or different tissues, such as tracheae and bronchial epithelium, should be carefully evaluated in further studies where respiratory tissues from the same location should be compared with the same inhibitory substances under comparable experimental settings.

It must also be noted that in clinical practice, vasopressin is mainly administered with norepinephrine to treat vasodilatory shocks. Our experiments examined both substances separately to elucidate their substance-specific signal transduction pathways. However, when co-administered, their additive effects on the PTV appear conclusive at the respiratory epithelium because norepinephrine and vasopressin both target highly specific receptors and ultimately lead to Ca²⁺ release from the ER via different, substance-specific mechanisms.

All three vasopressors examined in our experiments showed a significantly reduced maximum PTV when administered in a Ca²⁺-free buffer solution. The maximum PTV was also significantly reduced with dopamine in a Ca²⁺-free buffer solution, but the numerical decline was low, questioning the clinical relevance of that finding. The PTV amplitude markedly declined with norepinephrine and vasopressin, suggesting greater relevance. Furthermore, no increase in the PTV was observed after administering the three substances when intracellular caffeine sensitive Ca²⁺ stores were depleted. Therefore, we conclude that norepinephrine, vasopressin, and, to a lesser extent, dopamine use extracellular Ca²⁺ influx to ultimately affect the PTV.

Extracellular Ca²⁺ influx occurs via store-operated Ca²⁺ entry (SOCE), which is triggered by the release of Ca²⁺ from intracellular stores (Spinelli and Trebak, 2016). SOCE is modulated by different receptors and channels, such as the ORAI calcium release-activated calcium channels (Nguyen et al., 2018). The ORAI channels are activated by stromal interacting molecules located in the transmembrane area of the ER that sense decreased Ca²⁺ concentration in the ER (Umemura et al., 2023). Future studies should conduct further experiments evaluating the value of SOCE in tracheal epithelial cells, which could use isolated tracheal epithelial cells with calcium fluorescence measurements. While specific inhibitory substances of SOCE should be used, specific inhibition of distinct channels is difficult in the face of multiple potential targets and complex interactions (Spinelli and Trebak, 2016). Therefore, we decided to prevent overall extracellular Ca²⁺ entry using a Ca²⁺-free buffer solution, and these results should be considered hypothesis-generating.

Our study had the following limitations that must be acknowledged. Firstly, our experiments used murine tissues. Therefore, their results should be generalized cautiously to human tissues and patients. Using murine tissues in our experiments is a generally valid approach to evaluate hypotheses that can later be proven in experiments with human tissues. However, some differences might be significant even between different mammalians. For example, while the V_1b receptor seems to provoke the increase in the CBF in rabbit tissues, the V_1a receptor seems to cause these effects in murine tissues. Secondly, isolated tracheae were used in all experiments where subsequent intravenous drug administration was impossible, with the drugs administered directly into the buffer solution in which the tracheae were placed. However, the physiological integrity of the tracheae was preserved, and the epithelial barrier might have prevented drug entry from the apical side. Therefore, the concentration-response relationship might vary considerably in other tissues and clinical practice. Thirdly, the clearance function of tracheae might differ from the lower respiratory tracts, which primarily contribute to mucociliary clearance. Fourthly, the intravenous concentrations of the examined vasopressors are unknown in clinical practice and might even have significant interindividual differences, limiting the transferability of the concentrations evaluated in our experiments. Concentrations might also vary considerably between different tissues at different time points. For example, the examined vasopressors are mainly administered intravenously via central venous lines, although norepinephrine might also be delivered by inhalation. Therefore, regional concentrations might differ appreciably, especially in the pulmonary arteries and capillaries directly after central venous infusion. Finally, the sample size of our experimental groups was limited due to animal welfare regulations, which required us to use the smallest possible sample size.

In conclusion, our experiments showed that dopamine, norepinephrine, and vasopressin induce significant changes in the murine PTV. Our results suggest that they potentially modulate mucociliary clearance when applied in patients to obtain sufficient mean arterial blood pressure. Dopamine, norepinephrine, and vasopressin all initially act via substance-specific transmembrane receptors. However, they all ultimately lead to the release of intracellular Ca²⁺ from the ER, which is attenuated by additional extracellular Ca²⁺ influx. Further studies are needed to evaluate the clinical relevance of these findings and determine whether changes in mucociliary clearance could be proven in human tissues and patients during general anesthesia or critical illness when these vasopressors are used.