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Glucocorticoid decreases airway tone via a nongenomic pathway
Respiratory Physiology & Neurobiology 183 (2012) 10–14
Contents lists available at SciVerse ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Short communication
Glucocorticoid decreases airway tone via a nongenomic pathway Chen Wang a , Yi-Jia Li a , Yi-Qing Zheng a , Bing Feng a , Yan Liu b,∗∗ , Ji-Min Cao b,∗ a
Department of Medicine, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China b
a r t i c l e
i n f o
Article history: Accepted 7 May 2012 Keywords: Nocturnal asthma Circadian rhythm Airway tone Glucocorticoids Nongenomic effect
a b s t r a c t Nocturnal asthma is associated with circadian rhythms. Although glucocorticoids have contributed to therapeutic success, the underlying mechanism has not been studied thoroughly in asthma. Here, we report that cortisol, a member of glucocorticoids, ameliorate guinea pig tracheal spasm via a nongenomic effect. We set a concentration gradient of cortisol to mimic the functional circadian fluctuation. When administrated over a threshold (150 ng/ml), cortisol could synergize with the spasmolytic action of -agonist (isoprenaline) in histamine-sensitized tracheal spirals in vitro. This permissive action was abolished by the glucocorticoid receptor antagonist, RU486, indicating that cortisol acts via its receptor. Using the RNA polymerase inhibitor, actinomycin D, we showed that this permissive action was not affected by transcription. PMA, activator of protein kinase C (PKC), could partially imitate this rapid effect, while PKC inhibition also blocked this action to some extent. It is likely that this nongenomic effect of glucocorticoid underlies the onset and susceptibility of asthma, implying novel medication target in clinical practice. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nocturnal asthma is a kind of persistent asthma, associated with impaired life quality, high morbidity and mortality. It is defined as “diurnal changes of the forced expiratory volume in 1 s (FEV1) greater than 15% associated with increased airway hyperresponsiveness and inflammation” (Shigemitsu and Afshar, 2007). This disease follows the circadian rhythm and is associated with various mechanisms including inflammatory cells activity, cytokines, hormone concentration and hormone receptor affinity, etc. Given that the onsets of nocturnal asthma take place especially in the early morning (01:00–04:00), studies on chronopharmacotherapy is warranted to target medication at this specific period of time. To date, however, this predilection is still poorly addressed, and deeper insight into the mechanism of nocturnal asthma as well as chronopharmacotherapy is needed. Glucocorticoid, an essential hormone with its circulatory level following the circadian rhythm, has been postulated to be crucial in nocturnal asthma (Barnes et al., 1980). As the corner-stone for clinical management of inflammatory diseases in decades, previous studies mainly focus on its genomic pathway, suggesting that glucocorticoid targets inflammation via the intracellular glucocorticoid receptor (GR) (Hayashi et al., 2004). The genomic
∗ Corresponding author. Tel.: +86 10 6529 6959; fax: +86 10 6529 6959. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Liu), [email protected] (J.-M. Cao). 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2012.05.008
effect of glucocorticoid is known to occur with a time lag of hours or even days. Research has also identified some stressmediated responses of glucocorticoid that are much more rapid and take place in seconds or minutes. Rapid glucocorticoid actions are triggered by, or at least dependent on, membrane associated G protein-coupled receptors (GPCRs) and activation of downstream signaling cascades, although the molecular identities of these GPCRs are still elusive (Tasker et al., 2006). Particularly, glucocorticoids could also exert its effect on airway smooth muscle (ASM) via a nongenomic pathway (Sun et al., 2006). This action might also be important from a clinical point of view, as glucocorticoids induce short-term amelioration for asthma, rather than its long-term effects as anti-inflammatory and immunosuppressant agent in some chronic diseases, such as rheumatoid arthritis. Sun et al. (2006) has shown that budesonide (a derivative of glucocorticoids) could exert spasmolytic effects on guinea pig airway smooth muscles independent of genomic pathway. Nonetheless, no mechanistic issue was addressed in their study. Meanwhile, the concentration of budesonide they used was much higher than physiologic values. By its nature, glucocorticoids are high lipophilic substances that tend to accumulate in plasma membrane, so nonspecific action at the membrane level should be considered when glucocorticoids were applied at rather high dosage. This nonspecificity also hampered the search for membrane receptors of glucocorticoids by biochemical methods. Downstream from the putative membrane receptors, various signaling pathways have been implicated in the rapid actions of glucocorticoids in different cell types, and Gq -phospholipase C (PLC)-protein kinase C (PKC) is one of them (Qiu et al., 1998). The role of glucocorticoids at
C. Wang et al. / Respiratory Physiology & Neurobiology 183 (2012) 10–14
physiologic levels, the circadian rhythm and downstream signaling cascades of glucocorticoids remain largely uncharacterized in asthma-related models, such as sensitized ASM. Moreover, the genetic contribution of related genes in asthma is still under debate. We hypothesize that the nongenomic action of glucocorticoids is involved in airway tone regulation and asthma pathogenesis, and a threshold concentration is needed for glucocorticoid to exert its “permissive action” on the trachea-relaxing effect of -agonists. To address this hypothesis, we differentiated the potential nongenomic effect of glucocorticoid on airway tone using an ex vivo model of guinea pig tracheal smooth muscles which were pre-sensitized by histamine. Particularly, a concentration gradient of cortisol was set to recapitulate the functional circadian fluctuation, with an attention paid to identify the threshold cortisol concentration by which the permissive action was realized. The study may help to justify the importance of chronopharmacotherapy in treating nocturnal asthma. In addition, literature-based analysis suggested a genetic connection between multiple genes in the nongenomic pathway of glucocorticoid and asthma in human. These results provide a novel mechanistic explanation for how this pathway might influence the susceptibility and treatment of asthma, shedding light on antiasthmatic drug development. 2. Materials and methods 2.1. Animals Young Hartley guinea pigs (250–350 g) with no restriction to gender were used. The animals were housed under room temperature (22–26 ◦ C) and maintained in a 12:12 light–dark cycle. Regular food and water were free access. The animal use protocol was approved by the Life Ethics Committee of Peking Union Medical College and was conducted in compliance with the U.S. National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication 85-23). 2.2. Reagents Reagents were purchased from the following suppliers: cortisol (Tianjin Jinyao Amino Acid Co. Ltd., Tianjin, China), histamine (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), isoprenaline (Shanghai Harvest Pharmaceuticals Co. Ltd., Shanghai, China), RU486 (Beijing Zizhu Pharmaceutical Co. Ltd., Beijing, China), actinomycin D (Beyotime, Jiangsu, China), phorbol-12myristate-13-acetate (PMA) (Beyotime, Jiangsu, China), staurosporine C (Beyotime, Jiangsu, China). 2.3. Simulation of circadian rhythms by setting a glucocorticoid gradient Variations in glucocorticoid level is a systemic time cue in the body fluid and can be even used to elicit circadian gene expression in culture cells (Balsalobre et al., 2000). Given the well-controlled environment in ex vivo model, glucocorticoid can be applied in a dose-manner to mimic the circadian rhythm (DeRijk et al., 1997). In guinea pig, the physiological range of cortisol is from 64 ng/ml (in dark phase) to 123 ng/ml (peak, between 04:00 and 08:00 h) (Garris, 1979). However, other reports indicated that the range could be 70–300 ng/ml (Dalle and Delost, 1974). Therefore, we set the concentration gradient of cortisol as 50, 100, 150 and 200 ng/ml to mimic the physiological fluctuation of this major circadian hormone in regular light–dark cycle.
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2.4. Measurement of tracheal smooth muscle tension The animals were sacrificed by cervical dislocation, and the trachea was removed as soon as possible under room light during light phase. Isolated trachea was sunk in prewarmed Krebs–Henseleit (K-H) buffer solution (37 ◦ C), and the connective tissues were eliminated. Tracheal spiral was prepared as described previously (Drazen and Schneider, 1978). The tracheal spiral was put into a tissue chamber and perfused with K-H buffer solution containing (in mM): NaCl 118, KCl 4.7, MgSO4 ·7H2 O 3.4, CaCl2 ·2H2 O 2.52, NaHCO3 24.48, KH2 PO4 1.18, glucose 11, oxygenated continuously at 37 ◦ C. Before experiment, the smooth muscle spiral was preloaded with a 1 g tension initially and then stabilized for approximately 30 min. During stabilization period, the spiral was washed every 10 min. Histamine (10 M), coincident with a peak in early morning, was used to contract the tracheal smooth muscles. Relaxation was induced by -agonist, isoprenaline (0.1 M). Mechanical tension was recorded by the BL-420E data acquisition system through a force transducer (Chengdu Technology & Market Co. Ltd., Chengdu, China). 2.5. Therapeutic index calculation Therapeutic index is defined as “b/a”, which has been reported previously as “percentage inhibition of maximal contraction” (Chu et al., 2007). In this formula, “a” indicates maximal contraction induced by histamine while “b” refers to relaxation induced by agonist (Fig. 1A). The therapeutic value was normalized with the control group in each experimental set to eliminate the differences in responsiveness. 2.6. Statistical analysis All data were expressed in mean ± standard error (SEM). Statistics was analyzed by Student’s t-test and multiple comparisons were made by one-way analysis of variance (ANOVA). A p value less than 0.05 was considered statistically significant. 3. Results and discussion 3.1. Cortisol exerts its permissive action only when over a threshold, but itself has no impact on the muscle-constricting effect of histamine We first determined whether cortisol concentration impacted the efficacy of isoprenaline by setting a cortisol concentration gradient according to physiological cortisol levels of guinea pig. When cortisol level was above 150 ng/ml, still within the physiological range, the relaxation effect (Fig. 1B) and therapeutic index (Fig. 1C) of isoprenaline rose significantly compared with the baseline (p = 0.002). By contrast, other concentrations lower than 150 ng/ml did not show significant permissive action (Fig. 1B). This result suggests that cortisol synergizes with isoprenaline only when its concentration is above a threshold. To exclude the influence of cortisol on histamine-induced airway smooth muscle constriction, we assessed the contractile response to histamine at variable cortisol concentrations. Similar contractile responses were elicited on the tracheal smooth muscle strips that were pre-exposed to cortisol ranging from 50 to 200 ng/ml (Fig. 1D). In addition, the contractile response was constant when the cortisol was applied after histamine sensitization (Fig. 1B). In the clinical scenario, this result also suggests that the efficacy of -agonist at 3–4 a.m. is suboptimal due to low level of glucocorticoid at this specific period of time.
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C. Wang et al. / Respiratory Physiology & Neurobiology 183 (2012) 10–14
Fig. 1. Cortisol synergizes with the spasmolytic action of isoprenaline when over 150 ng/ml. (A) A schematic record of the tension curve. Cortisol, as a major circadian substance, was added to asthma stimulation system when contraction reached the plateau level. (B) The relaxation effect and (C) therapeutic index of isoprenaline at different cortisol concentrations. (D) Contractile responses to histamine at different cortisol concentrations. Dash lines indicate the plateau level of contraction. The concentration gradient of cortisol ranged from 0, 50, 100, 150 to 200 ng/ml. The way to calculate therapeutic index and normalization were described in Section 2. **p < 0.002 when 200 ng/ml cortisol group is compared with baseline group (n = 5 in each group).
3.2. PKC is involved in the nongenomic action of cortisol on airway tone Next, we tended to understand how cortisol exerts its synergistic effects, the so-called permissive action (Durant et al., 1983). Glucocorticoid receptor (GR), the effector of cortisol, might play an important role to both genomic and nongenomic pathways of glucocorticoids (Muto et al., 2000). After administration of RU486, a GR antagonist, the permissive action of cortisol was abolished (Fig. 2). Therefore, binding to GR is essential for the action of cortisol, indicating the critical role of GR in this process. Given that cortisol only works during a short period of time, it is not likely that genomic pathway accounts for the elevation of therapeutic index. In order to assess the importance of genomic pathway to this permissive action, we utilized actinomycin D to block all the transcription. After administration of actinomycin D, therapeutic index did not change significantly (p = 0.330) (Fig. 2). This result indicates that the well characterized genomic pathway does not lead to amelioration of therapeutic effects in our model. In comparison to the findings of Sun et al., however, ours suggest that GR is still essential to the nongenomic effect. A possible explanation to this discrepancy could be that we used physiologic levels
Fig. 2. Therapeutic index showing that cortisol synergizes with isoprenaline via a nongenomic pathway. Histamine sensitized tracheal spirals were treated with drugs, then mechanical tension was recorded and managed as described in the text. GR antagonist (RU486), RNA polymerase inhibitor (actinomycin D) and PKC inhibitor (staurosporine C) were given 20 min before cortisol treatment. PKC activator (PMA) was used in parallel with cortisol. Data represent the therapeutic index over basal values. **p < 0.002, *p < 0.05 vs. the control group, respectively (n = 3 in each group). Act, actinomycin D. STA, staurosporine C.
C. Wang et al. / Respiratory Physiology & Neurobiology 183 (2012) 10–14
of glucocorticoid, which is more relevant to the clinical scenario, while they used high levels of budesonide (10−5 mol/l) in comparison with ours (5 × 10−7 mol/l). Besides, the interaction of cytosolic receptors with other signaling systems has been suggested as an explanation for the nongenomic effect of steroid hormones (Lösel and Wehling, 2003). After demonstrating the irrelevance of the genomic pathway in this permissive action, we wanted to probe further the role of the nongenomic pathway in this process. Previous reports have suggested that glucocorticoid may exert short-term, nongenomic effect via protein kinase C (PKC) in vascular smooth muscle cells (Muto et al., 2000) and neuroblastoma cells (Han et al., 2002). In their research, short-term exposure to phorbol 12-myristate 13acetate (PMA), a PKC activator, mimicked the nongenomic effect of glucocorticoid. In order to see if activation of PKC pathway is able to imitate the effect of cortisol in our model, PMA was administered parallel to cortisol. Surprisingly, PMA did not fully mimic the permissive action of cortisol, given that mean therapeutic effect of 200 ng/l cortisol mounted to 1.29, while that of PMA was only 1.15 (Fig. 2). On the other hand, PKC inhibitor staurosporine C also partially abolished the permissive action (Fig. 2). This set of experiments indicates that PKC pathway only partially accounts for the elevation of therapeutic index; other pathways might also be involved in this process. Technically, these could also result from the activation profile of PMA, which did not fully recapitulate the effect of cortisol on PKC, due to binding affinity, exposure time or both. 3.3. The nongenomic pathway of cortisol is associated with asthma in human Finally, we tried to assess the clinical relevance of the nongenomic effect of glucocorticoid in human diseases, especially asthma. Since the wide application of glucocorticoids and its derivatives in asthma therapy, the genetic association of glucocorticoid receptor (NR3C1) has been a long-term target of researchers, particularly for the population with severe, difficult-to-treat asthma. Surprisingly, no association of glucocorticoid receptor (NR3C1) polymorphism has been observed in the 12 genome-wide association studies (GWAS) to look for susceptible loci for asthma and related traits (Akhabir and Sandford, 2011). However, we noticed the controversy in literatures. A polymorphism of NR3C1 was confirmed in a recent study of patients with bronchial asthma (Pietras et al., 2011). This polymorphism may represent the variant with low minor allele frequency, even the rare variant, which was largely missed by GWAS, but does contribute significantly to the heritability of complex diseases, herein asthma (Manolio et al., 2009). The association of NR3C1 can only be distinguished out for certain types of asthma, which was diluted in GWAS. We next asked whether there is a connection between PKC, the master mediator of nongenomic effect of glucocorticoids, and asthma susceptibility. As a major component of signal transduction, it is hard to imagine the role of PKC in asthma. To our surprise, PRKCA was mapped to the shared genetic region between asthma and obesity in children (Melén et al., 2010). Moreover, at least one single nucleotide polymorphism (SNP) (rs11079657) remains significant after adjustment for multiple comparisons (Murphy et al., 2009). Thus PRKCA was suggested as a pleiotropic locus that is associated with both BMI and asthma. Our findings stress the crucial role of nongenomic glucocorticoid pathway in the treatment of nocturnal asthma. Moreover, the aforementioned genetic associations emphasized the importance of glucocorticoid signaling in the susceptibility and treatment of asthma, especially the potential of glucocorticoid nongenomic effect. Intensive pursuing glucocorticoid with stronger anti-inflammatory effect has been conducted in industry. However,
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the serious side effects also limit their application. A better understanding of the molecular mode of glucocorticoids will result in identification of novel drugs with improved benefit–risk ratio, and molecules mimic the permissive action of glucocorticoids might emerge as such a candidate of antiasthmatic drugs, especially in the combinational application with 2-adrenergic agonists, for example, salmeterol. Further investigation is needed to elucidate the precise molecular mechanisms of this permissive action and define the downstream targets of this pathway. 4. Conclusions In conclusion, our studies have suggested that physiologic levels of glucocorticoid can synergize with -agonist in a permissive action. This effect depends on the nongenomic pathway of glucocorticoid involving PKC. From the perspective of clinical practice, our research suggests that 2-agonists might be more suboptimal than we have expected due to the poor permissive action of glucocorticoid at this specific period of time. Financial support This study was supported by grants of Scientific Research and Entrepreneurship for Undergraduates in Beijing City, and a grant (81071072 to J.-M.C.) from the Natural Science Foundation of China (NSFC). Conflict of interest None. Author contributions The experiment was designed and performed by undergraduate students (C. Wang, Y.-J. Li, Y.-Q. Zheng and B. Feng) during their course of physiological experiments. Y. Liu and J.-M. Cao are professors responsible for this course. Acknowledgement We thank Dr. S. Lei for technical support. References Akhabir, L., Sandford, A.J., 2011. Genome-wide association studies for discovery of genes involved in asthma. Respirology 16, 396–406. Balsalobre, A., Brown, S.A., Marcacci, L., Tronche, F., Kellendonk, C., Reichardt, H.M., Schütz, G., Schibler, U., 2000. Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289, 2344–2347. Barnes, P., FitzGerald, G., Brown, M., Dollery, C., 1980. Nocturnal asthma and changes in circulating epinephrine, histamine, and cortisol. New England Journal of Medicine 303, 263–267. Chu, X., Xu, Z., Wu, D., Zhao, A., Zhou, M., Qiu, M., Jia, W., 2007. In vitro and in vivo evaluation of the anti-asthmatic activities of fractions from Pheretima. Journal of Ethnopharmacology 111, 490–495. Dalle, M., Delost, P., 1974. Changes in the concentrations of cortisol and corticosterone in the plasma and adrenal glands of the guinea-pig from birth to weaning. Journal of Endocrinology 63, 483–488. DeRijk, R., Michelson, D., Karp, B., Petrides, J., Galliven, E., Deuster, P., Paciotti, G., Gold, P.W., Sternberg, E.M., 1997. Exercise and circadian rhythm-induced variations in plasma cortisol differentially regulate interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) production in humans: high sensitivity of TNF alpha and resistance of IL-6. Journal of Clinical Endocrinology and Metabolism 82, 2182–2191. Drazen, J.M., Schneider, M.W., 1978. Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro. Journal of Clinical Investigation 61, 1441–1447. Durant, S., Duval, D., Homo-Delarche, F., 1983. Potentiation by steroids of the beta-adrenergic agent-induced stimulation of cyclic AMP in isolated mouse thymocytes. Biochimica et Biophysica Acta 762, 315–324. Garris, D.R., 1979. Diurnal fluctuation of plasma cortisol levels in the guinea pig. Acta Endocrinology (Copenhagen) 90, 692–695.
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Han, J.Z., Lin, W., Lou, S.J., Qiu, J., Chen, Y.Z., 2002. A rapid, nongenomic action of glucocorticoids in rat B103 neuroblastoma cells. Biochimica et Biophysica Acta 1591, 21–27. Hayashi, R., Wada, H., Ito, K., Adcock, I.M., 2004. Effects of glucocorticoids on gene transcription. European Journal of Pharmacology 500, 51–62. Lösel, R., Wehling, M., 2003. Nongenomic actions of steroid hormones. Nature Reviews Molecular Cell Biology 4, 46–56. Manolio, T.A., Collins, F.S., Cox, N.J., Goldstein, D.B., Hindorff, L.A., Hunter, D.J., McCarthy, M.I., Ramos, E.M., Cardon, L.R., Chakravarti, A., Cho, J.H., Guttmacher, A.E., Kong, A., Kruglyak, L., Mardis, E., Rotimi, C.N., Slatkin, M., Valle, D., Whittemore, A.S., Boehnke, M., Clark, A.G., Eichler, E.E., Gibson, G., Haines, J.L., Mackay, T.F., McCarroll, S.A., Visscher, P.M., 2009. Finding the missing heritability of complex diseases. Nature 461, 747–753. Melén, E., Himes, B.E., Brehm, J.M., Boutaoui, N., Klanderman, B.J., Sylvia, J.S., LaskySu, J., 2010. Analyses of shared genetic factors between asthma and obesity in children. Journal of Allergy and Clinical Immunology 126, 631–637. Murphy, A., Tantisira, K.G., Soto-Quirós, M.E., Avila, L., Klanderman, B.J., Lake, S., Weiss, S.T., Celedón, J.C., 2009. PRKCA: a positional candidate gene for body mass index and asthma. American Journal of Human Genetics 85, 87–96.
Muto, S., Ebata, S., Okada, K., Saito, T., Asano, Y., 2000. Glucocorticoid modulates Na+ /H+ exchange activity in vascular smooth muscle cells by nongenomic and genomic mechanisms. Kidney International 57, 2319–2333. Pietras, T., Panek, M., Tworek, D., Oszajca, K., Wujcik, R., Górski, P., Kuna, P., Szemraj, J., 2011. The Bcl I single nucleotide polymorphism of the human glucocorticoid receptor gene h-GR/NR3C1 promoter in patients with bronchial asthma: pilot study. Molecular Biology Reports 38, 3953–3958. Qiu, J., Lou, L.G., Huang, X.Y., Lou, S.J., Pei, G., Chen, Y.Z., 1998. Nongenomic mechanisms of glucocorticoid inhibition of nicotine-induced calcium influx in PC12 cells: involvement of protein kinase C. Endocrinology 139, 5103–5108. Shigemitsu, H., Afshar, K., 2007. Nocturnal asthma. Current Opinion in Pulmonary Medicine 13, 49–55. Sun, H.W., Miao, C.Y., Liu, L., Zhou, J., Su, D.F., Wang, Y.X., Jiang, C.L., 2006. Rapid inhibitory effect of glucocorticoids on airway smooth muscle contractions in guinea pigs. Steroids 71, 154–159. Tasker, J.G., Di, S., Malcher-Lopes, R., 2006. Minireview: rapid glucocorticoid signaling via membrane-associated receptors. Endocrinology 147, 5549–5556.
Contents lists available at SciVerse ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Short communication
Glucocorticoid decreases airway tone via a nongenomic pathway Chen Wang a , Yi-Jia Li a , Yi-Qing Zheng a , Bing Feng a , Yan Liu b,∗∗ , Ji-Min Cao b,∗ a
Department of Medicine, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China b
a r t i c l e
i n f o
Article history: Accepted 7 May 2012 Keywords: Nocturnal asthma Circadian rhythm Airway tone Glucocorticoids Nongenomic effect
a b s t r a c t Nocturnal asthma is associated with circadian rhythms. Although glucocorticoids have contributed to therapeutic success, the underlying mechanism has not been studied thoroughly in asthma. Here, we report that cortisol, a member of glucocorticoids, ameliorate guinea pig tracheal spasm via a nongenomic effect. We set a concentration gradient of cortisol to mimic the functional circadian fluctuation. When administrated over a threshold (150 ng/ml), cortisol could synergize with the spasmolytic action of -agonist (isoprenaline) in histamine-sensitized tracheal spirals in vitro. This permissive action was abolished by the glucocorticoid receptor antagonist, RU486, indicating that cortisol acts via its receptor. Using the RNA polymerase inhibitor, actinomycin D, we showed that this permissive action was not affected by transcription. PMA, activator of protein kinase C (PKC), could partially imitate this rapid effect, while PKC inhibition also blocked this action to some extent. It is likely that this nongenomic effect of glucocorticoid underlies the onset and susceptibility of asthma, implying novel medication target in clinical practice. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nocturnal asthma is a kind of persistent asthma, associated with impaired life quality, high morbidity and mortality. It is defined as “diurnal changes of the forced expiratory volume in 1 s (FEV1) greater than 15% associated with increased airway hyperresponsiveness and inflammation” (Shigemitsu and Afshar, 2007). This disease follows the circadian rhythm and is associated with various mechanisms including inflammatory cells activity, cytokines, hormone concentration and hormone receptor affinity, etc. Given that the onsets of nocturnal asthma take place especially in the early morning (01:00–04:00), studies on chronopharmacotherapy is warranted to target medication at this specific period of time. To date, however, this predilection is still poorly addressed, and deeper insight into the mechanism of nocturnal asthma as well as chronopharmacotherapy is needed. Glucocorticoid, an essential hormone with its circulatory level following the circadian rhythm, has been postulated to be crucial in nocturnal asthma (Barnes et al., 1980). As the corner-stone for clinical management of inflammatory diseases in decades, previous studies mainly focus on its genomic pathway, suggesting that glucocorticoid targets inflammation via the intracellular glucocorticoid receptor (GR) (Hayashi et al., 2004). The genomic
∗ Corresponding author. Tel.: +86 10 6529 6959; fax: +86 10 6529 6959. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Liu), [email protected] (J.-M. Cao). 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2012.05.008
effect of glucocorticoid is known to occur with a time lag of hours or even days. Research has also identified some stressmediated responses of glucocorticoid that are much more rapid and take place in seconds or minutes. Rapid glucocorticoid actions are triggered by, or at least dependent on, membrane associated G protein-coupled receptors (GPCRs) and activation of downstream signaling cascades, although the molecular identities of these GPCRs are still elusive (Tasker et al., 2006). Particularly, glucocorticoids could also exert its effect on airway smooth muscle (ASM) via a nongenomic pathway (Sun et al., 2006). This action might also be important from a clinical point of view, as glucocorticoids induce short-term amelioration for asthma, rather than its long-term effects as anti-inflammatory and immunosuppressant agent in some chronic diseases, such as rheumatoid arthritis. Sun et al. (2006) has shown that budesonide (a derivative of glucocorticoids) could exert spasmolytic effects on guinea pig airway smooth muscles independent of genomic pathway. Nonetheless, no mechanistic issue was addressed in their study. Meanwhile, the concentration of budesonide they used was much higher than physiologic values. By its nature, glucocorticoids are high lipophilic substances that tend to accumulate in plasma membrane, so nonspecific action at the membrane level should be considered when glucocorticoids were applied at rather high dosage. This nonspecificity also hampered the search for membrane receptors of glucocorticoids by biochemical methods. Downstream from the putative membrane receptors, various signaling pathways have been implicated in the rapid actions of glucocorticoids in different cell types, and Gq -phospholipase C (PLC)-protein kinase C (PKC) is one of them (Qiu et al., 1998). The role of glucocorticoids at
C. Wang et al. / Respiratory Physiology & Neurobiology 183 (2012) 10–14
physiologic levels, the circadian rhythm and downstream signaling cascades of glucocorticoids remain largely uncharacterized in asthma-related models, such as sensitized ASM. Moreover, the genetic contribution of related genes in asthma is still under debate. We hypothesize that the nongenomic action of glucocorticoids is involved in airway tone regulation and asthma pathogenesis, and a threshold concentration is needed for glucocorticoid to exert its “permissive action” on the trachea-relaxing effect of -agonists. To address this hypothesis, we differentiated the potential nongenomic effect of glucocorticoid on airway tone using an ex vivo model of guinea pig tracheal smooth muscles which were pre-sensitized by histamine. Particularly, a concentration gradient of cortisol was set to recapitulate the functional circadian fluctuation, with an attention paid to identify the threshold cortisol concentration by which the permissive action was realized. The study may help to justify the importance of chronopharmacotherapy in treating nocturnal asthma. In addition, literature-based analysis suggested a genetic connection between multiple genes in the nongenomic pathway of glucocorticoid and asthma in human. These results provide a novel mechanistic explanation for how this pathway might influence the susceptibility and treatment of asthma, shedding light on antiasthmatic drug development. 2. Materials and methods 2.1. Animals Young Hartley guinea pigs (250–350 g) with no restriction to gender were used. The animals were housed under room temperature (22–26 ◦ C) and maintained in a 12:12 light–dark cycle. Regular food and water were free access. The animal use protocol was approved by the Life Ethics Committee of Peking Union Medical College and was conducted in compliance with the U.S. National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication 85-23). 2.2. Reagents Reagents were purchased from the following suppliers: cortisol (Tianjin Jinyao Amino Acid Co. Ltd., Tianjin, China), histamine (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), isoprenaline (Shanghai Harvest Pharmaceuticals Co. Ltd., Shanghai, China), RU486 (Beijing Zizhu Pharmaceutical Co. Ltd., Beijing, China), actinomycin D (Beyotime, Jiangsu, China), phorbol-12myristate-13-acetate (PMA) (Beyotime, Jiangsu, China), staurosporine C (Beyotime, Jiangsu, China). 2.3. Simulation of circadian rhythms by setting a glucocorticoid gradient Variations in glucocorticoid level is a systemic time cue in the body fluid and can be even used to elicit circadian gene expression in culture cells (Balsalobre et al., 2000). Given the well-controlled environment in ex vivo model, glucocorticoid can be applied in a dose-manner to mimic the circadian rhythm (DeRijk et al., 1997). In guinea pig, the physiological range of cortisol is from 64 ng/ml (in dark phase) to 123 ng/ml (peak, between 04:00 and 08:00 h) (Garris, 1979). However, other reports indicated that the range could be 70–300 ng/ml (Dalle and Delost, 1974). Therefore, we set the concentration gradient of cortisol as 50, 100, 150 and 200 ng/ml to mimic the physiological fluctuation of this major circadian hormone in regular light–dark cycle.
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2.4. Measurement of tracheal smooth muscle tension The animals were sacrificed by cervical dislocation, and the trachea was removed as soon as possible under room light during light phase. Isolated trachea was sunk in prewarmed Krebs–Henseleit (K-H) buffer solution (37 ◦ C), and the connective tissues were eliminated. Tracheal spiral was prepared as described previously (Drazen and Schneider, 1978). The tracheal spiral was put into a tissue chamber and perfused with K-H buffer solution containing (in mM): NaCl 118, KCl 4.7, MgSO4 ·7H2 O 3.4, CaCl2 ·2H2 O 2.52, NaHCO3 24.48, KH2 PO4 1.18, glucose 11, oxygenated continuously at 37 ◦ C. Before experiment, the smooth muscle spiral was preloaded with a 1 g tension initially and then stabilized for approximately 30 min. During stabilization period, the spiral was washed every 10 min. Histamine (10 M), coincident with a peak in early morning, was used to contract the tracheal smooth muscles. Relaxation was induced by -agonist, isoprenaline (0.1 M). Mechanical tension was recorded by the BL-420E data acquisition system through a force transducer (Chengdu Technology & Market Co. Ltd., Chengdu, China). 2.5. Therapeutic index calculation Therapeutic index is defined as “b/a”, which has been reported previously as “percentage inhibition of maximal contraction” (Chu et al., 2007). In this formula, “a” indicates maximal contraction induced by histamine while “b” refers to relaxation induced by agonist (Fig. 1A). The therapeutic value was normalized with the control group in each experimental set to eliminate the differences in responsiveness. 2.6. Statistical analysis All data were expressed in mean ± standard error (SEM). Statistics was analyzed by Student’s t-test and multiple comparisons were made by one-way analysis of variance (ANOVA). A p value less than 0.05 was considered statistically significant. 3. Results and discussion 3.1. Cortisol exerts its permissive action only when over a threshold, but itself has no impact on the muscle-constricting effect of histamine We first determined whether cortisol concentration impacted the efficacy of isoprenaline by setting a cortisol concentration gradient according to physiological cortisol levels of guinea pig. When cortisol level was above 150 ng/ml, still within the physiological range, the relaxation effect (Fig. 1B) and therapeutic index (Fig. 1C) of isoprenaline rose significantly compared with the baseline (p = 0.002). By contrast, other concentrations lower than 150 ng/ml did not show significant permissive action (Fig. 1B). This result suggests that cortisol synergizes with isoprenaline only when its concentration is above a threshold. To exclude the influence of cortisol on histamine-induced airway smooth muscle constriction, we assessed the contractile response to histamine at variable cortisol concentrations. Similar contractile responses were elicited on the tracheal smooth muscle strips that were pre-exposed to cortisol ranging from 50 to 200 ng/ml (Fig. 1D). In addition, the contractile response was constant when the cortisol was applied after histamine sensitization (Fig. 1B). In the clinical scenario, this result also suggests that the efficacy of -agonist at 3–4 a.m. is suboptimal due to low level of glucocorticoid at this specific period of time.
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Fig. 1. Cortisol synergizes with the spasmolytic action of isoprenaline when over 150 ng/ml. (A) A schematic record of the tension curve. Cortisol, as a major circadian substance, was added to asthma stimulation system when contraction reached the plateau level. (B) The relaxation effect and (C) therapeutic index of isoprenaline at different cortisol concentrations. (D) Contractile responses to histamine at different cortisol concentrations. Dash lines indicate the plateau level of contraction. The concentration gradient of cortisol ranged from 0, 50, 100, 150 to 200 ng/ml. The way to calculate therapeutic index and normalization were described in Section 2. **p < 0.002 when 200 ng/ml cortisol group is compared with baseline group (n = 5 in each group).
3.2. PKC is involved in the nongenomic action of cortisol on airway tone Next, we tended to understand how cortisol exerts its synergistic effects, the so-called permissive action (Durant et al., 1983). Glucocorticoid receptor (GR), the effector of cortisol, might play an important role to both genomic and nongenomic pathways of glucocorticoids (Muto et al., 2000). After administration of RU486, a GR antagonist, the permissive action of cortisol was abolished (Fig. 2). Therefore, binding to GR is essential for the action of cortisol, indicating the critical role of GR in this process. Given that cortisol only works during a short period of time, it is not likely that genomic pathway accounts for the elevation of therapeutic index. In order to assess the importance of genomic pathway to this permissive action, we utilized actinomycin D to block all the transcription. After administration of actinomycin D, therapeutic index did not change significantly (p = 0.330) (Fig. 2). This result indicates that the well characterized genomic pathway does not lead to amelioration of therapeutic effects in our model. In comparison to the findings of Sun et al., however, ours suggest that GR is still essential to the nongenomic effect. A possible explanation to this discrepancy could be that we used physiologic levels
Fig. 2. Therapeutic index showing that cortisol synergizes with isoprenaline via a nongenomic pathway. Histamine sensitized tracheal spirals were treated with drugs, then mechanical tension was recorded and managed as described in the text. GR antagonist (RU486), RNA polymerase inhibitor (actinomycin D) and PKC inhibitor (staurosporine C) were given 20 min before cortisol treatment. PKC activator (PMA) was used in parallel with cortisol. Data represent the therapeutic index over basal values. **p < 0.002, *p < 0.05 vs. the control group, respectively (n = 3 in each group). Act, actinomycin D. STA, staurosporine C.
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of glucocorticoid, which is more relevant to the clinical scenario, while they used high levels of budesonide (10−5 mol/l) in comparison with ours (5 × 10−7 mol/l). Besides, the interaction of cytosolic receptors with other signaling systems has been suggested as an explanation for the nongenomic effect of steroid hormones (Lösel and Wehling, 2003). After demonstrating the irrelevance of the genomic pathway in this permissive action, we wanted to probe further the role of the nongenomic pathway in this process. Previous reports have suggested that glucocorticoid may exert short-term, nongenomic effect via protein kinase C (PKC) in vascular smooth muscle cells (Muto et al., 2000) and neuroblastoma cells (Han et al., 2002). In their research, short-term exposure to phorbol 12-myristate 13acetate (PMA), a PKC activator, mimicked the nongenomic effect of glucocorticoid. In order to see if activation of PKC pathway is able to imitate the effect of cortisol in our model, PMA was administered parallel to cortisol. Surprisingly, PMA did not fully mimic the permissive action of cortisol, given that mean therapeutic effect of 200 ng/l cortisol mounted to 1.29, while that of PMA was only 1.15 (Fig. 2). On the other hand, PKC inhibitor staurosporine C also partially abolished the permissive action (Fig. 2). This set of experiments indicates that PKC pathway only partially accounts for the elevation of therapeutic index; other pathways might also be involved in this process. Technically, these could also result from the activation profile of PMA, which did not fully recapitulate the effect of cortisol on PKC, due to binding affinity, exposure time or both. 3.3. The nongenomic pathway of cortisol is associated with asthma in human Finally, we tried to assess the clinical relevance of the nongenomic effect of glucocorticoid in human diseases, especially asthma. Since the wide application of glucocorticoids and its derivatives in asthma therapy, the genetic association of glucocorticoid receptor (NR3C1) has been a long-term target of researchers, particularly for the population with severe, difficult-to-treat asthma. Surprisingly, no association of glucocorticoid receptor (NR3C1) polymorphism has been observed in the 12 genome-wide association studies (GWAS) to look for susceptible loci for asthma and related traits (Akhabir and Sandford, 2011). However, we noticed the controversy in literatures. A polymorphism of NR3C1 was confirmed in a recent study of patients with bronchial asthma (Pietras et al., 2011). This polymorphism may represent the variant with low minor allele frequency, even the rare variant, which was largely missed by GWAS, but does contribute significantly to the heritability of complex diseases, herein asthma (Manolio et al., 2009). The association of NR3C1 can only be distinguished out for certain types of asthma, which was diluted in GWAS. We next asked whether there is a connection between PKC, the master mediator of nongenomic effect of glucocorticoids, and asthma susceptibility. As a major component of signal transduction, it is hard to imagine the role of PKC in asthma. To our surprise, PRKCA was mapped to the shared genetic region between asthma and obesity in children (Melén et al., 2010). Moreover, at least one single nucleotide polymorphism (SNP) (rs11079657) remains significant after adjustment for multiple comparisons (Murphy et al., 2009). Thus PRKCA was suggested as a pleiotropic locus that is associated with both BMI and asthma. Our findings stress the crucial role of nongenomic glucocorticoid pathway in the treatment of nocturnal asthma. Moreover, the aforementioned genetic associations emphasized the importance of glucocorticoid signaling in the susceptibility and treatment of asthma, especially the potential of glucocorticoid nongenomic effect. Intensive pursuing glucocorticoid with stronger anti-inflammatory effect has been conducted in industry. However,
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the serious side effects also limit their application. A better understanding of the molecular mode of glucocorticoids will result in identification of novel drugs with improved benefit–risk ratio, and molecules mimic the permissive action of glucocorticoids might emerge as such a candidate of antiasthmatic drugs, especially in the combinational application with 2-adrenergic agonists, for example, salmeterol. Further investigation is needed to elucidate the precise molecular mechanisms of this permissive action and define the downstream targets of this pathway. 4. Conclusions In conclusion, our studies have suggested that physiologic levels of glucocorticoid can synergize with -agonist in a permissive action. This effect depends on the nongenomic pathway of glucocorticoid involving PKC. From the perspective of clinical practice, our research suggests that 2-agonists might be more suboptimal than we have expected due to the poor permissive action of glucocorticoid at this specific period of time. Financial support This study was supported by grants of Scientific Research and Entrepreneurship for Undergraduates in Beijing City, and a grant (81071072 to J.-M.C.) from the Natural Science Foundation of China (NSFC). Conflict of interest None. Author contributions The experiment was designed and performed by undergraduate students (C. Wang, Y.-J. Li, Y.-Q. Zheng and B. Feng) during their course of physiological experiments. Y. Liu and J.-M. Cao are professors responsible for this course. Acknowledgement We thank Dr. S. Lei for technical support. References Akhabir, L., Sandford, A.J., 2011. Genome-wide association studies for discovery of genes involved in asthma. Respirology 16, 396–406. Balsalobre, A., Brown, S.A., Marcacci, L., Tronche, F., Kellendonk, C., Reichardt, H.M., Schütz, G., Schibler, U., 2000. Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289, 2344–2347. Barnes, P., FitzGerald, G., Brown, M., Dollery, C., 1980. Nocturnal asthma and changes in circulating epinephrine, histamine, and cortisol. New England Journal of Medicine 303, 263–267. Chu, X., Xu, Z., Wu, D., Zhao, A., Zhou, M., Qiu, M., Jia, W., 2007. In vitro and in vivo evaluation of the anti-asthmatic activities of fractions from Pheretima. Journal of Ethnopharmacology 111, 490–495. Dalle, M., Delost, P., 1974. Changes in the concentrations of cortisol and corticosterone in the plasma and adrenal glands of the guinea-pig from birth to weaning. Journal of Endocrinology 63, 483–488. DeRijk, R., Michelson, D., Karp, B., Petrides, J., Galliven, E., Deuster, P., Paciotti, G., Gold, P.W., Sternberg, E.M., 1997. Exercise and circadian rhythm-induced variations in plasma cortisol differentially regulate interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) production in humans: high sensitivity of TNF alpha and resistance of IL-6. Journal of Clinical Endocrinology and Metabolism 82, 2182–2191. Drazen, J.M., Schneider, M.W., 1978. Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro. Journal of Clinical Investigation 61, 1441–1447. Durant, S., Duval, D., Homo-Delarche, F., 1983. Potentiation by steroids of the beta-adrenergic agent-induced stimulation of cyclic AMP in isolated mouse thymocytes. Biochimica et Biophysica Acta 762, 315–324. Garris, D.R., 1979. Diurnal fluctuation of plasma cortisol levels in the guinea pig. Acta Endocrinology (Copenhagen) 90, 692–695.
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