Estrogen effects on human airway smooth muscle involve camp and protein kinase A
|
|
- Godwin Noel Phelps
- 6 years ago
- Views:
Transcription
1 Am J Physiol Lung Cell Mol Physiol 303: L923 L928, First published September 21, 2012; doi: /ajplung Estrogen effects on human airway smooth muscle involve camp and protein kinase A Elizabeth A. Townsend, 1,3 Venkatachalem Sathish, 1,2 Michael A. Thompson, 2 Christina M. Pabelick, 1,2 and Y. S. Prakash 1,2 Departments of 1 Physiology and Biomedical Engineering and 2 Anesthesiology, Mayo Clinic, Rochester, Minnesota; 3 Department of Anesthesiology, Columbia University Medical Center, New York, New York Submitted 13 January 2012; accepted in final form 18 September 2012 Townsend EA, Sathish V, Thompson MA, Pabelick CM, Prakash YS. Estrogen effects on human airway smooth muscle involve camp and protein kinase A. Am J Physiol Lung Cell Mol Physiol 303: L923 L928, First published September 21, 2012; doi: /ajplung Clinically observed differences in airway reactivity and asthma exacerbations in women at different life stages suggest a role for sex steroids in modulating airway function although their targets and mechanisms of action are still being explored. We have previously shown that clinically relevant concentrations of exogenous estrogen acutely decrease intracellular calcium ([Ca 2 ] i ) in human airway smooth muscle (ASM), thereby facilitating bronchodilation. In this study, we hypothesized that estrogens modulate cyclic nucleotide regulation, resulting in decreased [Ca 2 ] i in human ASM. In Fura-2-loaded human ASM cells, 1 nm 17 -estradiol (E 2 ) potentiated the inhibitory effect of the -adrenoceptor ( -AR) agonist isoproterenol (ISO; 100 nm) on histamine-mediated Ca 2 entry. Inhibition of protein kinase A (PKA) activity (KT5720; 100 nm) attenuated E 2 effects on [Ca 2 ] i. Acute treatment with E 2 increased camp levels in ASM cells comparable to that of ISO (100 pm). In acetylcholine-contracted airways from female guinea pigs or female humans, E 2 potentiated ISO-induced relaxation. These novel data suggest that, in human ASM, physiologically relevant concentrations of estrogens act via estrogen receptors (ERs) and the camp pathway to nongenomically reduce [Ca 2 ] i, thus promoting bronchodilation. Activation of ERs may be a novel adjunct therapeutic avenue in reactive airway diseases in combination with established campactivating therapies such as 2 -agonists. sex steroid; bronchial smooth muscle; bronchodilation; bronchoconstriction; calcium; asthma; -adrenoceptor ASTHMA, A RESPIRATORY DISEASE characterized by airway inflammation and hyperresponsiveness, is more common in adult women compared with men (9, 24, 27, 32). Furthermore, adult women experience changes in symptom severity and the number of exacerbations with changes in hormonal status (different phases of the menstrual cycle, pregnancy, and menopause) (1, 6, 7, 16, 18). Thus clinical evidence suggests a role for sex steroids (especially female sex steroids) in modulating airway reactivity. What is less clear is whether estrogens or progesterone are detrimental or beneficial to the airway. Whereas clinical evidence would suggest the former, whole animal studies have found reduced airway hyperresponsiveness in the absence of estrogens or estrogen receptor (ER) signaling (3, 4, 10, 23). We previously demonstrated that, in human airway smooth muscle (ASM) cells, clinically relevant concentrations of 17 -estradiol (E 2 ) reduce intracellular Ca 2 concentration Address for reprint requests and other correspondence: Y. S. Prakash, Dept. of Anesthesiology, W Joseph SMH, Mayo Clinic, 200 1st St. SW, Rochester MN ( prakash.ys@mayo.edu). ([Ca 2 ] i ) (32). Thus it is possible that estrogens promote bronchodilation by acting on ASM. -2 Adrenoceptor ( 2 -AR) agonists are the most effective bronchodilators available to date and are a mainstay of treatment for acute asthma exacerbations (13, 22). Although 2 - agonists are generally effective, tachyphylaxis with repeated use as well as reduced efficacy are well-recognized limitations. Accordingly, there is considerable interest in development of adjunct and novel approaches to enhancing bronchodilation. Drugs that activate 2 -AR are known to increase adenylyl cyclase (AC) activity and adenosine 3=,5=-cyclic monophosphate (camp), activate protein kinase A (PKA), and produce downstream effects including inhibition of Ca 2 influx, acceleration of sarcoplasmic reticulum Ca 2 reuptake, and reduction of myosin light chain kinase activity. Studies in nonairway tissues have demonstrated that E 2 can increase camp. For example, E 2 -induced protein kinase G-dependent relaxation in the cardiovascular system is mediated by camp (19, 20). Vasodilation of porcine coronary arteries by E 2 also occurs via camp in an endothelium-independent manner (31) (although others have reported that E 2 does not affect camp levels; 8). Although the mechanisms of E 2 effects in the airway are still under investigation, these data raise the question of whether estrogens can increase camp in ASM and contribute to bronchodilation. Here, an additional point of interest would be whether estrogens, by working through a beneficial mechanism common to 2 -AR, could be used as an adjunct. In this study, we investigated the mechanisms of rapid, nongenomic actions of estrogen in human ASM, with the hypothesis that E 2 increases camp and PKA activity. We previously showed that E 2 greatly attenuates Ca 2 influx, in part by acting on L-type Ca 2 channels (32), which are also a target of 2 -AR agonists (5). Accordingly, we tested the hypothesis that signaling overlap exists between acute estrogen signaling and 2 -AR signaling in human ASM. MATERIALS AND METHODS Isolation of human ASM cells. Studies were conducted using ASM cells enzymatically dissociated from lower-order bronchi (3rd to 6th generation) from lung samples incidental to patient (female only) surgery at Mayo Clinic, Rochester (deidentified samples; typically from lobectomies and pneumonectomies; approved and considered exempt by Mayo Institutional Review Board). Visibly normal airway, as confirmed by a pathologist s review, was excised and rapidly transferred to the laboratory in ice-cold Hanks Balanced Salt Solution (HBSS). ASM cells were enzymatically dissociated as previously described (32) using a papain dissociation kit (Worthington Biochemical, Lakewood, NJ). Cells were maintained at 37 C (5% CO 2-95% air) using phenol red-free DMEM/F12 medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum until 80% conflu /12 Copyright 2012 the American Physiological Society L923
2 L924 ent. Phenol red-free media was used to avoid ER activation. All experiments were performed in cells from passages 1 3 of subculture that had been serum starved for 48 h before experimentation. Periodic assessment of ASM phenotype was performed by verifying stable expression of smooth muscle actin and myosin, and agonist receptors, with lack of fibroblast marker expression. camp assay. ASM cells grown to 80% confluence were nonenzymatically harvested and centrifuged at 200 g for 2 min, washed with PBS, and recentrifuged. The cell pellet was resuspended in HBSS containing 5 mm HEPES and 0.5 mm IBMX, ph 7.4. This cell suspension was then treated according to manufacturer s instructions (LANCE Ultra camp Kit, Perkin-Elmer, Beverly, MA). The cell suspension (5 l containing 1,000 cells) was added to 5 l of agonist solutions and allowed to incubate for 30 min at room temperature in an OptiPlate 384-well plate. The detection mix containing Eu-cAMP tracer and ULight-anti-cAMP was added and allowed to incubate for 1 h. The assay was read on a Molecular Devices Flexstation 3 system (LANCE settings; 340 nm Ex/665 nm Em; Sunnyvale, CA). Cyclic AMP standards included with the kit and cell suspensions stimulated with HBSS (containing IBMX) only served as standard curve and internal controls, respectively. Western blot analysis. Standard immunoblotting techniques were used for detection of ER (SC-542; Santa Cruz Biotechnology, Santa Cruz, CA), ER (Santa Cruz SC-53494), and 2-AR (Santa Cruz SC-9042) and detected using horseradish peroxidase-conjugated secondary antibodies in ASM cell lysates subjected to coimmunoprecipitation. Primary antibody (2 g, rabbit anti- 2-AR, Santa Cruz SC- 9042) was used per 200 l of whole cell lysate and incubated overnight at 4 C with gentle rotation. Protein A agarose beads (50 l) were added to the sample and incubated for 4 h at 4 C. Proteins were recovered as described previously (32). These samples were then processed as described for Western analysis ( 2-AR; no ; Sigma-Aldrich, St. Louis, MO). Blots were imaged on a Kodak ImageStation 4000mm (Carestream Health, New Haven, CT) and quantified using densitometry. [Ca 2 ]i imaging. Techniques for real-time Ca 2 imaging have been previously described (29, 32). Briefly, ASM cells were loaded with the fluorescent Ca 2 indicator Fura-2 AM (5 M, 50 min, room temperature) and imaged using a Metafluor-based real-time microscopy system (Nikon Instruments TE2000 inverted microscope; 40/ 1.3 NA oil-immersion lens; 1 Hz; acquisition of 510 nm emissions following alternative excitation at 340 vs. 380 nm). In all experiments involving [Ca 2 ] i measurements, 10 M histamine was used as an agonist to induce [Ca 2 ] i increases. Force measurements. All animal experiments were approved by Columbia University s Animal Care and Use Committee, and animal care was in accordance with the guidelines published by the American Physiological Society. Female Hartley guinea pigs ( 400 g) were obtained from Charles River and were euthanized with intraperitoneal injection of pentobarbital. The trachea was quickly removed and placed on ice in Krebs-Hensleit buffer of the following composition in mm: 118 NaCl, 5.6 KCl, 0.5 CaCl 2, 0.24 MgSO 4, 1.3 NaH 2PO 4,25 NaHCO 3, and 5.6 glucose, ph 7.4. The epithelium was removed by gentle abrasion with cotton. Detailed methods have been previously described (14). Briefly, tracheal rings were suspended in 4-ml-water jacketed organ baths (37 C: Radnoti Glass Technology, Monrovia, CA) and connected to a Grass FT03 force transducer (Grass Telefactor, West Warwick, RI) using silk sutures and adjusted to a resting tension of 1 g and allowed to equilibrate for 1 h with buffer exchanges every 15 min. Two complete acetylcholine (ACh) dose-response curves were constructed (100 nm-100 M) and then thoroughly washed. Rings were then contracted with EC 50 ACh. Following stable force generation with ACh, samples were treated with vehicle or 50 nm E 2 for 20 min, followed by an isoproterenol (ISO) dose response curve (100 pm-10 M; half-log increments). Measurements are expressed as a percentage of remaining contraction of AChplateau force. Parallel studies were conducted in isolated, epithelium-denuded bronchial rings from female patients, obtained as described above for cells. To avoid confounding effects of variable chronic inflammation and/or cigarette smoke exposure, medical histories were reviewed, and samples were taken only from female patients without these problems. Materials. 2=,5=-Dideoxyadenosine was obtained from Tocris Biosciences (Ellisville, MO). E 2, ISO, KT5720, forskolin, and other chemicals were obtained from Sigma Chemical unless mentioned otherwise. Tissue culture reagents, including Dulbecco s Modified Eagle s Medium F-12 (DMEM F/12) and FBS, were obtained from Invitrogen. Statistical analysis. Seven bronchial samples from different female patients were used to obtain ASM cells. [Ca 2 ] i experiments were performed in at least 25 cells, each from four different bronchial (patient) samples although not all protocols were performed in each sample obtained. In all cases using ASM cells, n represents numbers of patients. The guinea pig trachea studies involved five animals with eight rings/animal. In experiments where responses were compared in the presence or absence of specific drugs, paired t-test was used, whereas population studies were compared using unpaired t-test or one-way ANOVA with repeated measures as appropriate. Bonferroni correction was applied for multiple comparisons. Statistical significance was established at P All values are expressed as means SE. RESULTS Estrogen potentiates ISO effects on [Ca 2 ] i regulation. Ca 2 imaging of Fura-2-loaded ASM cells showed that exposure to E 2 (1 nm) for 15 min significantly reduced by 40% increases in peak [Ca 2 ] i induced by histamine, consistent with our previous finding (32) (Fig. 1A; P 0.05). Exposure for 15 min to 100 nm ISO also significantly reduced [Ca 2 ] i responses to histamine to comparable levels (Fig. 1A; P 0.05). The additional presence of E 2 (1 nm) during ISO exposure resulted in a larger, 65% reduction in peak [Ca 2 ] i, which was significantly different from either E 2 or ISO alone (Fig. 1, A and C; n 5, P 0.05 compared with vehicle as well as to E 2 ). Estrogen effects on ASM involve adenylyl cyclase. In a separate set of experiments, ASM cells were pretreated with the AC inhibitor, 2=,5=-dideoxyadenosine (20 M) for 15 min before and during exposure to E 2, ISO, or a combination of E 2 and ISO (concentrations as above). Cells were then stimulated with 10 M histamine. The AC inhibitor completely prevented the previously observed reduction of [Ca 2 ] i responses induced by ISO (Fig. 1, B and C: n 5; P 0.05 for inhibitor effect). However, a residual 20 25% reduction in [Ca 2 ] i responses persisted in groups treated with E 2 (Fig. 1, B and C; P 0.05). ER activation increases camp. In a first set of experiments, baseline camp accumulation by human ASM cells was evaluated using increasing concentrations of forskolin (1 nm to 100 M, half-log increments) and time-resolved fluorescence resonance energy transfer (TR-FRET). Cyclic AMP standards were used to construct a TR-FRET-concentration curve and calculate the amount of camp (Fig. 2A). Baseline camp by the forskolin method showed a dynamic range of 30 nm to 10 M (Fig. 2B). Subsequently, the effects of E 2 (10 nm), ISO (100 pm), or the combination of E 2 and ISO were evaluated. All three treatments showed a substantial increase in camp compared with vehicle (HBSS only) controls (Fig. 2C; n 5 8, *P 0.05). The E 2 and ISO combination showed an additive effect in total camp produced (Fig. 2C; n 5 8, #P 0.05 compared with E 2 or ISO alone). It should be noted
3 L925 KT5720 (100 nm) for 15 min before and during E 2 treatment completely prevented E 2 effects on the [Ca 2 ] i responses to histamine. KT5720 treatment alone (i.e., no E 2 ) did not substantially alter the [Ca 2 ] i response to histamine (Fig. 3: n 4, P 0.05). Estrogen potentiates relaxation effects of isoproterenol on contracted airways. In epithelium-denuded tracheal rings from female guinea pigs contracted with 1 M ACh ( EC 50 based on a control dose-response curve), the presence of 50 nm E 2 increased the extent of relaxation induced by ISO (100 pm-100 Downloaded from Fig. 1. A: estradiol (E 2) potentiates -agonist effects on airway smooth muscle (ASM) intracellular calcium concentration ([Ca 2 ] i). In Fura-2-loaded human ASM cells, histamine-induced increases in [Ca 2 ] i were blunted by isoproterenol (ISO; 100 nm) and E 2 (1 nm). The combination of E 2 and ISO further decreased [Ca 2 ] i response to agonist (a representative tracing of 5 experiments). B: inhibition of adenylyl cyclase blunts E 2 effects. In Fura-2-loaded cells, the effect of ISO was reversed with the adenylyl cyclase (AC) inhibitor 2=,5=-dideoxyadenosine (20 M 15 min alone, 15 min ISO). Treatment with this inhibitor partially reverses the observed E 2 effects (a representative tracing of 5 experiments). The combination of ISO and E 2 was reversed to the same levels as E 2 alone yet remained significant compared with vehicle (n 5). C: summary of estradiol effects on calcium regulation in ASM. E 2 reduced [Ca 2 ] i response to histamine by 45%. ISO decreased [Ca 2 ] i by 50%. The combination of ISO and E 2 had a greater effect than either treatment alone, reducing [Ca 2 ] i by 60%. ISO, the combination of ISO and E 2 and E 2 alone were reversed by AC inhibition. *P 0.05 significant compared with vehicle. $P 0.05 significant compared with ISO or E 2 alone. #P 0.05 significant inhibitor effect compared with ISO, E 2, and ISO E 2, respectively (n 5). that lower concentration of ISO was used in this analysis based on pilot studies showing saturation of the camp assay when 100 nm concentrations were used. Estrogen effects on ASM are PKA dependent. In Fura-2- loaded ASM cells, pretreatment with the PKA antagonist Fig. 2. camp regulation in ASM cells. A: cyclic-amp standards (10 pm-1 M; half-log increments) were used to create a concentration calibration based on decreasing time-resolved fluorescent resonance energy transfer (TR-FRET). B: in ASM cells, forskolin (1 nm-100 M; half-log increments) caused a concentration-dependent decrease in TR-FRET and thus increase in camp. Dose response curve of mean values (line 4 parameter sigmoid fit R ) C: in ASM cells, 100 pm ISO substantially increased camp over basal camp in untreated controls. Treatment with 10 nm E 2 increased camp to levels comparable to ISO. Treatment combining E 2 and ISO further increased camp above basal levels *P 0.05 significant compared with vehicle. #P 0.05 compared with E 2 or ISO alone (n 5 8). by on June 24, 2017
4 L926 Fig. 3. A and B: E 2 effects on ASM involve PKA activation. Acute exposure to E 2 decreases [Ca 2 ] i response to 10 M histamine by 40%. This response is abrogated by pretreatment with the PKA antagonist KT5720 (100 nm, 15 min alone, 15 min E 2). KT5720 alone had no effect on [Ca 2 ] i responses to histamine compared with vehicle. *P 0.05 significant compared with vehicle, #P 0.05 significant KT5720 compared with E 2 alone (n 4). nm, half-log increments) compared with vehicle-treated tissues (Fig. 4A; P 0.05 for E 2 effect). Indeed, in the presence of E 2, relaxation to and below baseline was achieved at lower ISO concentrations (Fig. 4A). This was also reflected by greater maximum relaxation of % in the presence of E 2 vs % with vehicle, when additional ISO concentrations were added (Fig. 4A, n 40 rings from 5 animals, *P 0.001, paired t-test). We compared the efficacy of E 2 to enhance ISO-induced relaxation in epithelium-denuded human bronchial rings. Complete relaxation was observed at 10 nm in the absence of E 2, which was comparable to that observed in the guinea pig (not shown). Furthermore, in the presence of E 2, the extent of relaxation and sensitivity to ISO were increased, such that, at 300 pm ISO, the human bronchial rings showed greater relaxation in the presence of E 2 (Fig. 4B; P 0.05). ERs colocalize with 2 -AR in human ASM. Immunoprecipitation (IP) of the 2 -AR with subsequent Western analysis for ER- or ER- revealed that both ER isoforms colocalize with 2 -AR in separate IPs (Fig. 5). A strong band at 66 kda corresponding to the full-length ER- was visible, whereas a band of slightly lower molecular weight (56 kda) corresponding to ER- was also expressed. Band intensity varied between patients and was proportional to protein concentration ( , ER- ; , ER- ; Fig. 5). Fig. 4. E 2 potentiates ISO-induced relaxation of ASM. A: in epitheliumdenuded bronchial rings from female guinea pigs contracted with acetylcholine (ACh, 1 M EC 50), pretreatment with 50 nm E 2 significantly increased relaxation to ISO (100 pm-10 M; half-log increments), with maximal relaxation below baseline also being greater in the presence of E 2 (n 40 rings from 5 animals). B: as with guinea pig, relaxation of ACh precontracted epithelium-denuded human bronchial rings induced by 300 pm ISO was enhanced by E 2.*P 0.05 significant E 2 effect (n 5). DISCUSSION We have previously demonstrated that clinically relevant concentrations of estrogens reduce [Ca 2 ] i in human ASM (32).The present study now demonstrates the role of camp and Fig. 5. Estrogen receptor (ER)- and ER- colocalize with 2-adrenergic receptor (AR) in human ASM. Coimmunoprecipitation of 2-ARs and subsequent Western analyses revealed that ER-, and, in separate experiment, ER- colocalizes with 2-AR (n 3 patients). Although band intensity varied between patients, expression of both ER- and ER- was proportional to protein concentration.
5 PKA in mediating these effects. The role of the camp/pka pathway in 2 -AR agonist action in bronchodilation is well recognized, as are tolerance and 2 -AR desensitization with chronic use (2). Importantly, our results show that estrogens can potentiate the effects of isoproterenol in reducing [Ca 2 ] i in human ASM via activation of camp and PKA. This potentiating effect appears to translate to enhanced bronchodilation, evidenced by increased ISO-induced relaxation of bronchial rings from both guinea pigs and humans when E 2 is present. Here, the colocalization of 2 -AR and ER may play a role in the interaction between these receptors in potentiating relaxation. Our data hint at a potentially novel avenue for enhancing 2 -AR-induced bronchodilation by modulation of estrogen signaling. Given the common, camp-dependent mechanism for both receptors, the relevance of such an avenue lies in the possibility of reducing 2 -AR utilization and thus desensitization. Estrogen signaling in the airway. Sex differences in airway function of women at different life stages have raised the question of hormonal influences, especially on ASM, the major regulator of airway tone. In this regard, the limited data on sex steroid effects on airway function are conflicting. Clinical data would suggest a worsening of airway function with increased estrogen levels. However, from an in vivo perspective, ER- deficient mice exhibit increased airway hyperresponsiveness (4), an effect reversed by hormone replacement in ovariectomized animals (10), consistent with a bronchodilatory role for estrogens. In vitro, we previously showed that both full-length ER- and ER- (32) are present in ASM of female patients and that clinically relevant concentrations of estrogens can activate these receptors to reduce agonist-induced elevations in [Ca 2 ] i via inhibition of L-type Ca 2 channels and store-operated Ca 2 entry (32). The in vitro cellular data suggesting a bronchodilatory effect of estrogens are generally consistent with data in mouse tracheal and bronchial rings sensitized by serum from asthmatic humans, where carbachol-induced contractions are reduced by 100 nm E 2 (11). These effects are also similar to those observed in the vasculature (26) and occur on a rapid time scale (as also demonstrated in the present study), distinct from the well-known transcriptional (genomic) response to estrogens (17). The reasons underlying the discrepancies between the clinical and preclinical data in estrogen effects are not clear but may involve the balance between genomic vs. nongenomic effects as well as estrogen effects on the airway vs. the immune system (30). Regardless, what is important to note is that, as with the vasculature, estrogens do have nongenomic effects on time scales similar to the activation patterns for the cyclic nucleotide cascades, which lend credence to the idea that estrogen effects on [Ca 2 ] i (and thus tone) may be mediated via camp. The present study demonstrates that estrogen activates the camp/pka pathway with downstream effects on [Ca 2 ] i and relaxation. Estrogen effects on camp and PKA signaling in other tissues are controversial. Studies in porcine coronary arteries show that E 2 either increases (19, 31) or has no effect on camp levels (8). In rat heart, Wong and colleagues (33) found that estrogen inhibits L-type Ca 2 channel influx, blocks isoprenaline-induced increase in camp, and suppresses PKA activity. However, in that study, these effects appear to involve estrogen-induced downregulation of 1 -AR. In the context of these previous studies in nonairway tissues, the results of the present study are novel in highlighting an estrogen-mediated elevation in camp and activation of PKA in human ASM. Under physiological conditions, it is likely that E 2 activates ER- and ER-, both of which were shown to independently colocalize with 2 -ARs in human ASM. The downstream effects of estrogen-induced activation of the camp/pka pathway is reduced [Ca 2 ] i in ASM cells and potentiation of ISO-induced relaxation of precontracted ASM tissue. The camp-pka pathway can certainly modulate a number of Ca 2 regulatory mechanisms. In neurons (25, 34), nongenomic, membrane actions of estrogen involve voltagegated calcium channels, which are also a target in ASM (32) and thus a potential mechanism for camp-induced reduction in [Ca 2 ] i. In a previous study also in human ASM, we have shown that store-operated Ca 2 entry, a mechanism inhibited by estrogens (32), is also inhibited by camp (28). PKA is also known to phosphorylate the Ca 2 -sensitive large conductance potassium (BK Ca ) channel (21), and previous studies in murine models of asthma have shown beneficial effects of estrogen being mediated in part via BK Ca channels (11); as such, it is likely that estrogen-induced increases in camp/pka activity affect plasma membrane Ca 2 regulatory mechanisms in ASM. Estrogen and 2 -AR effects in ASM. The role of the camp/ PKA pathway in 2 -AR action in ASM is well known (13, 15). It is also recognized that frequent or sustained 2 -AR activation results in receptor internalization and desensitization (2), raising the need for alternative or adjunct avenues to sustain bronchodilatory activity. The results of the present study show that clinically relevant concentrations of estrogen can potentiate 2 -AR induced reduction in [Ca 2 ] i of ASM, as well as relaxation of ASM. From a clinical perspective, there are limited data supporting a beneficial role for estrogens. For example, Gibbs and colleagues (12) showed that, in the few days before the onset of menses, women asthmatics had significantly decreased peak expiratory flow rates and increased dependence on albuterol (12), whereas exogenous estrogen has been reported to improve asthma symptoms in women (6). Importantly, our data show that acute estrogen administration potentiates the effects of isoproterenol, resulting in greater relaxation at submaximal isoproterenol concentrations as well as greater maximal relaxation compared with isoproterenoltreated tissues alone. Furthermore, we show that both estrogens and 2 -AR activate a common mechanism, camp/pka, thus raising the possibility of using estrogens to enhance 2 -AR agonist effects to induce bronchodilation. Whether the combination of estrogens and 2 -AR agonists is of benefit clinically remains to be shown. ACKNOWLEDGMENTS L927 The authors Dr. Charles Emala and Dr. Yi Zhang for help and input with these studies. GRANTS This work was supported by a Young Clinical Scientist Award from the Flight Attendants Medical Research Institute (FAMRI) to V. Sathish, and NIH R01 grants HL to Y. Prakash, HL to C. Pabelick, and T32GM to E. Townsend. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.
6 L928 REFERENCES 1. Bellia V, Augugliaro G. Asthma and menopause. Monaldi Arch Chest Dis 67: , Broadley KJ. Review of mechanisms involved in the apparent differential desensitization of 1- and 2-adrenoceptor-mediated functional responses. J Auton Pharmacol 19: , Card JW, Carey MA, Bradbury JA, DeGraff LM, Morgan DL, Moorman MP, Flake GP, Zeldin DC. Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation. J Immunol 177: , Carey MA, Card JW, Bradbury JA, Moorman MP, Haykal-Coates N, Gavett SH, Graves JP, Walker VR, Flake GP, Voltz JW, Zhu D, Jacobs ER, Dakhama A, Larsen GL, Loader JE, Gelfand EW, Germolec DR, Korach KS, Zeldin DC. Spontaneous airway hyperresponsiveness in estrogen receptor-alpha-deficient mice. Am J Respir Crit Care Med 175: , Catterall WA. Structure and regulation of voltage-gated Ca 2 channels. Annu Rev Cell Dev Biol 16: , Chandler MH, Schuldheisz S, Phillips BA, Muse KN. Premenstrual asthma: the effect of estrogen on symptoms, pulmonary function, and 2-receptors. Pharmacotherapy 17: , Chhabra SK. Premenstrual asthma. Indian J Chest Dis Allied Sci 47: , Christ M, Gunther A, Heck M, Schmidt BM, Falkenstein E, Wehling M. Aldosterone, not estradiol, is the physiological agonist for rapid increases in camp in vascular smooth muscle cells. Circulation 99: , de Marco R, Locatelli F, Sunyer J, Burney P. Differences in incidence of reported asthma related to age in men and women. A retrospective analysis of the data of the European Respiratory Health Survey. Am J Respir Crit Care Med 162: 68 74, Dimitropoulou C, Drakopanagiotakis F, Chatterjee A, Snead C, Catravas JD. Estrogen replacement therapy prevents airway dysfunction in a murine model of allergen-induced asthma. Lung 187: , Dimitropoulou C, White RE, Ownby DR, Catravas JD. Estrogen reduces carbachol-induced constriction of asthmatic airways by stimulating large-conductance voltage and calcium-dependent potassium channels. Am J Respir Cell Mol Biol 32: , Gibbs CJ, Coutts II, Lock R, Finnegan OC, White RJ. Premenstrual exacerbation of asthma. Thorax 39: , Giembycz MA, Newton R. Beyond the dogma: novel 2-adrenoceptor signalling in the airways. Eur Respir J 27: , Gleason NR, Gallos G, Zhang Y, Emala CW. The GABAA agonist muscimol attenuates induced airway constriction in guinea pigs in vivo. J Appl Physiol 106: , Hakonarson H, Grunstein MM. Regulation of second messengers associated with airway smooth muscle contraction and relaxation. Am J Respir Crit Care Med 158: S115 S122, Hanley SP. Asthma variation with menstruation. Br J Dis Chest 75: , Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague M, Strom A, Treuter E, Warner M, Gustafsson JA. Estrogen receptors: how do they signal and what are their targets. Physiol Rev 87: , Juniper EF, Daniel EE, Roberts RS, Kline PA, Hargreave FE, Newhouse MT. Effect of pregnancy on airway responsiveness and asthma severity. Relationship to serum progesterone. Am Rev Respir Dis 143: S78, Keung W, Chan ML, Ho EY, Vanhoutte PM, Man RY. Non-genomic activation of adenylyl cyclase and protein kinase G by 17 -estradiol in vascular smooth muscle of the rat superior mesenteric artery. Pharmacol Res 64: , Keung W, Vanhoutte PM, Man RY. Acute impairment of contractile responses by 17 -estradiol is camp and protein kinase G dependent in vascular smooth muscle cells of the porcine coronary arteries. Br J Pharmacol 144: 71 79, Kotlikoff MI, Kamm KE. Molecular mechanisms of -adrenergic relaxation of airway smooth muscle. Annu Rev Physiol 58: , Krymskaya VP, Panettieri RA Jr. Phosphodiesterases regulate airway smooth muscle function in health and disease. Curr Top Dev Biol 79: 61 74, Matsubara S, Swasey CH, Loader JE, Dakhama A, Joetham A, Ohnishi H, Balhorn A, Miyahara N, Takeda K, Gelfand EW. Estrogen determines sex differences in airway responsiveness after allergen exposure. Am J Respir Cell Mol Biol 38: , Melgert BN, Ray A, Hylkema MN, Timens W, Postma DS. Are there reasons why adult asthma is more common in females? Curr Allergy Asthma Rep 7: , Mermelstein PG, Becker JB, Surmeier DJ. Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor. J Neurosci 16: , Miller VM, Duckles SP. Vascular actions of estrogens: functional implications. Pharmacol Rev 60: , Paoletti P. Application of biomarkers in population studies for respiratory non-malignant diseases. Toxicology 101: , Prakash YS, Iyanoye A, Ay B, Sieck GC, Pabelick CM. Store-operated Ca 2 influx in airway smooth muscle: Interactions between volatile anesthetic and cyclic nucleotide effects. Anesthesiology 105: , Prakash YS, Thompson MA, Vaa B, Matabdin I, Peterson TE, He T, Pabelick CM. Caveolins and intracellular calcium regulation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 293: L1118 L1126, Straub RH. The complex role of estrogens in inflammation. Endocr Rev 28: , Teoh H, Man RY. Enhanced relaxation of porcine coronary arteries after acute exposure to a physiological level of 17 -estradiol involves nongenomic mechanisms and the cyclic AMP cascade. Br J Pharmacol 129: , Townsend EA, Thompson MA, Pabelick CM, Prakash YS. Rapid effects of estrogen on intracellular Ca 2 regulation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 298: L521 L530, Wong KA, Ma Y, Cheng WT, Wong TM. Cardioprotection by the female sex hormone interaction with the 1-adrenoceptor and its signaling pathways. Sheng Li Xue Bao 59: , Zhang C, Bosch MA, Rick EA, Kelly MJ, Ronnekleiv OK Estradiol regulation of T-type calcium channels in gonadotropin-releasing hormone neurons. J Neurosci 29: , 2009.
SUPPLEMENTAL MATERIAL. Supplementary Methods
SUPPLEMENTAL MATERIAL Supplementary Methods Culture of cardiomyocytes, fibroblasts and cardiac microvascular endothelial cells The isolation and culturing of neonatal rat ventricular cardiomyocytes was
More informationFor Research Use Only
LANCE Ultra camp Kit For Research Use Only 1. Intended use The LANCE Ultra camp kit is intended for the quantitative determination of 3,5 -cyclic monophosphate (camp) in cell culture and cellular membrane
More informationBitter Taste Receptors: Taking a Bigger Bite of Airway Smooth Muscle Pathophysiology
Page 1 of 6 Editorial Bitter Taste Receptors: Taking a Bigger Bite of Airway Smooth Muscle Pathophysiology Charles W. Emala, Sr. Department of Anesthesiology Columbia University, New York, New York Contact
More informationPrestretched airway smooth muscle response to length oscillation
ORIGINAL RESEARCH Physiological Reports ISSN 2051-817X Prestretched airway smooth muscle response to length oscillation Ahmed M. Al-Jumaily, Kevin Roos, Sandy Bessaguet & Miguel Jo Avila Institute of Biomedical
More informationTFEB-mediated increase in peripheral lysosomes regulates. Store Operated Calcium Entry
TFEB-mediated increase in peripheral lysosomes regulates Store Operated Calcium Entry Luigi Sbano, Massimo Bonora, Saverio Marchi, Federica Baldassari, Diego L. Medina, Andrea Ballabio, Carlotta Giorgi
More informationRelaxation responses of aortic rings from salt-loaded high calcium fed rats to potassium chloride, calcium chloride and magnesium sulphate
Pathophysiology 4 (1998) 275 280 Relaxation responses of aortic rings from salt-loaded high calcium fed rats to potassium chloride, calcium chloride and magnesium sulphate B.J. Adegunloye, O.A. Sofola
More informationSupplementary Material and Methods
Online Supplement Kockx et al, Secretion of Apolipoprotein E from Macrophages 1 Supplementary Material and Methods Cloning of ApoE-GFP Full-length human apoe3 cdna (pcdna3.1/zeo + -apoe) was kindly provided
More informationLearning Outcomes. Systems Pharmacology PHAR3320. Nerves of the Respiratory Tract. Dr Fernandes
Systems Pharmacology PHAR3320 Nerves of the Respiratory Tract Dr Fernandes Learning Outcomes By the end of this lecture, students should be able to describe nerve pathways that innervate the airways discuss
More informationRobert Kruklitis, MD, PhD Chief, Pulmonary Medicine Lehigh Valley Health Network
Robert Kruklitis, MD, PhD Chief, Pulmonary Medicine Lehigh Valley Health Network Robert.kruklitis@lvh.com Correlation of a Asthma pathophyisology with basic science Asthma (Physiology) Bronchodilators
More informationThe effect of L-arginine on guinea-pig and rabbit airway smooth muscle function in vitro
Brazilian Journal of Medical and Biological Research (1998) 31: 811-818 L-arginine on airway smooth muscle ISSN -879X 811 The effect of L-arginine on guinea-pig and rabbit airway smooth muscle function
More informationSoluble ADAM33 initiates airway remodeling to promote susceptibility for. Elizabeth R. Davies, Joanne F.C. Kelly, Peter H. Howarth, David I Wilson,
Revised Suppl. Data: Soluble ADAM33 1 Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life Elizabeth R. Davies, Joanne F.C. Kelly, Peter H. Howarth, David
More informationRapid inhibitory effect of glucocorticoids on airway smooth muscle contractions in guinea pigs
steroids 71 (2006) 154 159 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Rapid inhibitory effect of glucocorticoids on airway smooth muscle contractions in guinea
More informationINFLUENCE OF MENSTRUAL CYCLE ON LUNG FUNCTIONS IN ASTHMATIC FEMALES
INFLUENCE OF MENSTRUAL CYCLE ON LUNG FUNCTIONS IN ASTHMATIC FEMALES *Dimple Arora 1, Parminder Kaur 1, Mohit Arora 2 and Urmil Gupta 3 1 Department of Physiology, Sri Guru Ramdas Institute of Medical Sciences
More informationBCOR 011 Lecture 19 Oct 12, 2005 I. Cell Communication Signal Transduction Chapter 11
BCOR 011 Lecture 19 Oct 12, 2005 I. Cell Communication Signal Transduction Chapter 11 External signal is received and converted to another form to elicit a response 1 Lecture Outline 1. Types of intercellular
More informationDrug Receptor Interactions and Pharmacodynamics
Drug Receptor Interactions and Pharmacodynamics Dr. Raz Mohammed MSc Pharmacology School of Pharmacy 22.10.2017 Lec 6 Pharmacodynamics definition Pharmacodynamics describes the actions of a drug on the
More informationSodium Fluoride Improves Methacholine Induced Bronchoconstriction Similar To Salbutamol In Patients With Asthma
ISPUB.COM The Internet Journal of Asthma, Allergy and Immunology Volume 5 Number 1 Sodium Fluoride Improves Methacholine Induced Bronchoconstriction Similar To Salbutamol In Patients R Sonia, T Zouhair,
More informationRayBio KinaseSTAR TM Akt Activity Assay Kit
Activity Assay Kit User Manual Version 1.0 March 13, 2015 RayBio KinaseSTAR TM Akt Activity Kit Protocol (Cat#: 68AT-Akt-S40) RayBiotech, Inc. We Provide You With Excellent Support And Service Tel:(Toll
More informationValidation & Assay Performance Summary
Validation & Assay Performance Summary LanthaScreen IGF-1R GripTite Cells Cat. no. K1834 Modification Detected: Phosphorylation of Multiple Tyr Residues on IGF-1R LanthaScreen Cellular Assay Validation
More informationHawthorn Extract - Viable Treatment for Cardiovascular Disease or Unscrupulous Herbal Supplement?
Grand Valley State University ScholarWorks@GVSU Student Summer Scholars Undergraduate Research and Creative Practice 2010 Hawthorn Extract - Viable Treatment for Cardiovascular Disease or Unscrupulous
More informationAMPK Phosphorylation Assay Kit
AMPK Phosphorylation Assay Kit Catalog Number KA3789 100 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle
More informationHuman TRPC6 Ion Channel Cell Line
TECHNICAL DATA SHEET ValiScreen Ion Channel Cell Line Caution: For Laboratory Use. A research product for research purposes only Human TRPC6 Ion Channel Cell Line Product No.: AX-012-C Lot No.: 512-548-A
More informationPhysiology Unit 1 CELL SIGNALING: CHEMICAL MESSENGERS AND SIGNAL TRANSDUCTION PATHWAYS
Physiology Unit 1 CELL SIGNALING: CHEMICAL MESSENGERS AND SIGNAL TRANSDUCTION PATHWAYS In Physiology Today Cell Communication Homeostatic mechanisms maintain a normal balance of the body s internal environment
More informationFig. S1. High K+ increases intracellular calcium level.
Fig. S1. High K + increases intracellular calcium level. (A) Neuronal activation measured by calcium imaging using Fura-2. Intracellular calcium levels were continuously monitored by the fura-2 florescence
More informationNeurotransmitter Systems II Receptors. Reading: BCP Chapter 6
Neurotransmitter Systems II Receptors Reading: BCP Chapter 6 Neurotransmitter Systems Normal function of the human brain requires an orderly set of chemical reactions. Some of the most important chemical
More informationHistamine Develops Homologous Desensitization under Ca 2+ -free Conditions with Increase in Basal Tone in Smooth Muscle of Guinea Pig Taenia Caeci
YAKUGAKU ZASSHI 130(3) 451 455 (2010) 2010 The Pharmaceutical Society of Japan 451 Notes Histamine Develops Homologous Desensitization under Ca 2+ -free Conditions with Increase in Basal Tone in Smooth
More informationSupporting Information
Supporting Information Burford et al. 1.173/pnas.1339311 SI Materials and Methods β-arrestin Recruitment Assay. PathHunter human osteosarcoma cells (U2OS) expressing either μ-opioid receptors (U2OS- OPRM1)
More informationLecture 9: Cell Communication I
02.05.10 Lecture 9: Cell Communication I Multicellular organisms need to coordinate cellular functions in different tissues Cell-to-cell communication is also used by single celled organisms to signal
More informationReceptors Families. Assistant Prof. Dr. Najlaa Saadi PhD Pharmacology Faculty of Pharmacy University of Philadelphia
Receptors Families Assistant Prof. Dr. Najlaa Saadi PhD Pharmacology Faculty of Pharmacy University of Philadelphia Receptor Families 1. Ligand-gated ion channels 2. G protein coupled receptors 3. Enzyme-linked
More informationMTC-TT and TPC-1 cell lines were cultured in RPMI medium (Gibco, Breda, The Netherlands)
Supplemental data Materials and Methods Cell culture MTC-TT and TPC-1 cell lines were cultured in RPMI medium (Gibco, Breda, The Netherlands) supplemented with 15% or 10% (for TPC-1) fetal bovine serum
More informationEnhancement of synaptic transmission by cyclic AMP modulation of presynaptic I h channels. Vahri Beaumont and Robert S. Zucker
Enhancement of synaptic transmission by cyclic AMP modulation of presynaptic I h channels Vahri Beaumont and Robert S. Zucker Background I h channels discovered in 1976 (Noma A. and Irisawa H.) Voltage-gated
More informationQuercetin acutely relaxes airway smooth muscle and potentiates -agonist-induced relaxation via dual phosphodiesterase inhibition of PLC and PDE4
Am J Physiol Lung Cell Mol Physiol 305: L396 L403, 2013. First published July 19, 2013; doi:10.1152/ajplung.00125.2013. Quercetin acutely relaxes airway smooth muscle and potentiates -agonist-induced relaxation
More informationINVOLVEMENT OF K+ CHANNELS IN THE RELAXANT RESPONSES TO VARIOUS AGONISTS IN ESTROGEN PRIMED RAT UTERUS
ndian J Physiol Pharmacol 1995; 39(2) : 140-144 NVOLVEMENT OF K+ CHANNELS N THE RELAXANT RESPONSES TO VAROUS AGONSTS N ANTA A. MEHTA KRSHNA KANT C. DAVE* AND RAMESH K. GOYAL Department of Pharmacology
More informationRESPIRATORY PHARMACOLOGY - ASTHMA. Primary Exam Teaching - Westmead ED
RESPIRATORY PHARMACOLOGY - ASTHMA Primary Exam Teaching - Westmead ED Sympathomimetic agents MOA: relax airway smooth muscle and inhibit broncho constricting mediators from mast cells May also inhibit
More informationObjectives. Functions of smooth muscle. Smooth muscle. Smooth Muscle Contraction: Mechanism. Latch state. Smooth muscle contraction
Objectives Functions of smooth muscle Sompol Tapechum,, M.D., Ph.D. Department of Physiology Faculty of Medicine Siriraj hospital อธ บายล กษณะการหดต วของกล ามเน อเร ยบได อธ บายกลไกและป จจ ยท ม ผลต อการหดต
More informationLong- and short-acting β 2 adrenoceptor agonists: interactions in human contracted bronchi
Eur Respir J 1998; 11: 583 588 DOI: 1.1183/931936.98.113583 Printed in UK - all rights reserved Copyright ERS Journals Ltd 1998 European Respiratory Journal ISSN 93-1936 Long- and short-acting β 2 adrenoceptor
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION FOR Liver X Receptor α mediates hepatic triglyceride accumulation through upregulation of G0/G1 Switch Gene 2 (G0S2) expression I: SUPPLEMENTARY METHODS II: SUPPLEMENTARY FIGURES
More informationEffect of Prostaglandin F2ahjha n the Contractile Tissues of the Respiratory System of the Cat in Experimental Airway Inflammation
Physiol. Res. 40: 75-79, 1991 Effect of Prostaglandin F2ahjha n the Contractile Tissues of the Respiratory System of the Cat in Experimental Airway Inflammation p. b An o v Ci n, P. VISNOVSKY Department
More informationEFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL MIGRATION IN PANCREATIC CANCER CELLS
EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL MIGRATION IN PANCREATIC CANCER CELLS VIDYA MEDEPALLI ADVISOR: Maria Eugenia Sabbatini, PhD PHI KAPPA PHI RESEARCH CONFERENCE MARCH 8 th, 2017 Pancreatic
More informationApplication Note. Introduction
Simultaneously Measuring Oxidation of Exogenous and Endogenous Fatty Acids Using the XF Palmitate-BSA FAO Substrate with the Agilent Seahorse XF Cell Mito Stress Test Application Note Introduction The
More informationResults and discussion
Supplementary Figure 1. Assessment of the cross-talk between FuraPE3 and D4ER fluorescent spectra. Cells were perifused with 15 mmol/l glucose (G15) in the presence of 250 µmol/l of the KATP channel opener
More informationUse of a camp BRET Sensor to Characterize a Novel Regulation of camp by the Sphingosine-1-phosphate/G 13 Pathway
Use of a camp BRET Sensor to Characterize a Novel Regulation of camp by the Sphingosine-1-phosphate/G 13 Pathway SUPPLEMENTAL DATA Characterization of the CAMYEL sensor and calculation of intracellular
More informationDifferential responses to endothelial dependent relaxation of the thoracic and abdominal aorta from male Sprague-Dawley rats
Niger. J. Physiol. Sci. 27(December 12) 117 122 www.njps.com.ng Differential responses to endothelial dependent relaxation of the thoracic and abdominal aorta from male Sprague-Dawley rats 1 Oloyo, Ahmed
More informationThis laboratory exercise uses a simple preparation and a straightforward
LABORATORY DEMONSTRATION OF VASCULAR SMOOTH MUSCLE FUNCTION USING RAT AORTIC RING SEGMENTS Rayna J. Gonzales, Rebecca W. Carter, and Nancy L. Kanagy Vascular Physiology Group, Department of Cell Biology
More informationImprovements in Live Cell Analysis of G Protein Coupled Receptors using Second Generation BD Calcium Assay Kits
1 Current Chemical Genomics, 8, 2, 1-15 Open Access Improvements in Live Cell Analysis of G Protein Coupled Receptors using Second Generation BD Calcium Assay Kits Xiao Li*, Isabel Llorente and Mike Brasch
More informationSystems Pharmacology Respiratory Pharmacology. Lecture series : General outline
Systems Pharmacology 3320 2017 Respiratory Pharmacology Associate Professor Peter Henry (Rm 1.34) Peter.Henry@uwa.edu.au Division of Pharmacology, School of Biomedical Sciences Lecture series : General
More informationSupporting Information
Supporting Information Identification of Novel ROS Inducers: Quinone Derivatives Tethered to Long Hydrocarbon Chains Yeonsun Hong,, Sandip Sengupta,, Wooyoung Hur, *, Taebo Sim,, * KU-KIST Graduate School
More informationEFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL PROLIFERATION IN PANCREATIC CANCER CELLS
EFFECTORS IMPLICATED IN THE AC1 INHIBITORY EFFECT ON CELL PROLIFERATION IN PANCREATIC CANCER CELLS VIDYA MEDEPALLI ADVISOR: Maria Eugenia Sabbatini, PhD PHI KAPPA PHI RESEARCH CONFERENCE MARCH 18 TH, 2016
More informationAN INTRODUCTION TO INVOLUNTARY (ESPECIALLY SMOOTH) MUSCLES 1
AN INTRODUCTION TO INVOLUNTARY (ESPECIALLY SMOOTH) MUSCLES 1 Summary: This section is an introduction to a fascinating and extremely important group of tissue, the smooth muscles. As you will see, their
More informationA Hepatocyte Growth Factor Receptor (Met) Insulin Receptor hybrid governs hepatic glucose metabolism SUPPLEMENTARY FIGURES, LEGENDS AND METHODS
A Hepatocyte Growth Factor Receptor (Met) Insulin Receptor hybrid governs hepatic glucose metabolism Arlee Fafalios, Jihong Ma, Xinping Tan, John Stoops, Jianhua Luo, Marie C. DeFrances and Reza Zarnegar
More informationSupplementary Materials for
immunology.sciencemag.org/cgi/content/full/2/16/eaan6049/dc1 Supplementary Materials for Enzymatic synthesis of core 2 O-glycans governs the tissue-trafficking potential of memory CD8 + T cells Jossef
More informationEffect of cocaine on the affinity of a-adrenoceptors for noradrenaline
Br. J. Pharmac. (1973), 48, 139-143. Effect of cocaine on the affinity of a-adrenoceptors for noradrenaline I. R. INNES AND R. MAILHOT* Department of Pharmacology and Therapeutics, Faculty of Medicine,
More informationEffect of ageing on ƒ 1A-adrenoceptor mechanisms in rabbit. Issei TAKAYANAGI, Mann MORIYA and Katsuo KOIKE
J. Smooth Muscle Res. 28: 63-68, 1992. Effect of ageing on ƒ 1A-adrenoceptor mechanisms in rabbit isolated bronchial preparations Issei TAKAYANAGI, Mann MORIYA and Katsuo KOIKE Department of Chemical Pharmacology,
More informationSUPPLEMENTARY MATERIAL
SUPPLEMENTARY MATERIAL Table S1. Primers and fluorescent probes used for qrt-pcr analysis of relative expression levels of PPP family phosphatases. gene name forward primer, 5-3 probe, 5-3 reverse primer,
More informationThe pharmacogenetics of β 2 -adrenergic receptors: Relevance to asthma
The pharmacogenetics of β 2 -adrenergic receptors: Relevance to asthma Stephen B. Liggett, MD Cincinnati, Ohio The β 2 -adrenergic receptor (β 2 AR) is the molecular target for β-agonists used in the treatment
More information2401 : Anatomy/Physiology
Dr. Chris Doumen Week 11 2401 : Anatomy/Physiology Autonomic Nervous System TextBook Readings Pages 533 through 552 Make use of the figures in your textbook ; a picture is worth a thousand words! Work
More informationLujain Hamdan. Ayman Musleh & Yahya Salem. Mohammed khatatbeh
12 Lujain Hamdan Ayman Musleh & Yahya Salem Mohammed khatatbeh the last lecture, we have studied the differences between the two divisions of the ANS: sympathetic and parasympathetic pathways which work
More informationIII on the other hand.
Br. Br. J. J. Pharmacol. Pharmacol. (I (1993), 19, 774 774 778 778 '." Macmillan Press Ltd, 1993 Effects of rolipram and siguazodan on the human isolated bronchus and their interaction with isoprenaline
More informationDual-Controller Asthma Therapy: Rationale and Clinical Benefits
B/1 Dual-Controller Asthma Therapy: Rationale and Clinical Benefits MODULE B The 1997 National Heart, Lung, and Blood Institute (NHLBI) Expert Panel guidelines on asthma management recommend a 4-step approach
More informationThe dynamic regulation of blood vessel caliber
INVITED BASIC SCIENCE REVIEW The dynamic regulation of blood vessel caliber Colleen M. Brophy, MD, Augusta, Ga BACKGROUND The flow of blood to organs is regulated by changes in the diameter of the blood
More informationPHSI3009 Frontiers in Cellular Physiology 2017
Overview of PHSI3009 L2 Cell membrane and Principles of cell communication L3 Signalling via G protein-coupled receptor L4 Calcium Signalling L5 Signalling via Growth Factors L6 Signalling via small G-protein
More informationEpithelial interleukin-25 is a key mediator in Th2-high, corticosteroid-responsive
Online Data Supplement: Epithelial interleukin-25 is a key mediator in Th2-high, corticosteroid-responsive asthma Dan Cheng, Zheng Xue, Lingling Yi, Huimin Shi, Kan Zhang, Xiaorong Huo, Luke R. Bonser,
More informationReviewers' comments: Reviewer #1 Expert in EAE and IL-17a (Remarks to the Author):
Reviewers' comments: Reviewer #1 Expert in EAE and IL-17a (Remarks to the Author): This study shows that the inducible camp early repressor (ICER) is involved in development of Th17 cells that are pathogenic
More informationSUPPLEMENTARY INFORMATION
Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with
More informationRespiratory Pharmacology PCTH 400 Asthma and β-agonists
Respiratory Pharmacology PCTH 400 Asthma and β-agonists Dr. Tillie-Louise Hackett Department of Anesthesiology, Pharmacology and Therapeutics University of British Columbia Associate Director, Centre of
More informationEssential Medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Huvec were cultured in
Supplemental data Methods Cell culture media formulations A-431 and U-87 MG cells were maintained in Dulbecco s Modified Eagle s Medium. FaDu cells were cultured in Eagle's Minimum Essential Medium, containing
More informationXenoestrogen-induced Regulation of EZH2 and Histone Methylation via Non-Genomic Estrogen
Xenoestrogen-induced Regulation of EZH2 and Histone Methylation via Non-Genomic Estrogen Receptor Signaling to PI3K/AKT Tiffany G. Bredfeldt, Kristen L. Greathouse, Stephen H. Safe, Mien-Chie Hung, Mark
More informationInvestigation of the effects of vitamin D and calcium on intestinal motility: In vitro tests and implications for clinical treatment
Acta Pharm. 65 (2015) 343 349 DOI: 10.1515/acph-2015-0023 Short communication Investigation of the effects of vitamin D and calcium on intestinal motility: In vitro tests and implications for clinical
More informationBasics of Pharmacology
Basics of Pharmacology Pekka Rauhala Transmed 2013 What is pharmacology? Pharmacology may be defined as the study of the effects of drugs on the function of living systems Pharmacodynamics The mechanism(s)
More informationThe Schedule and the Manual of Basic Techniques for Cell Culture
The Schedule and the Manual of Basic Techniques for Cell Culture 1 Materials Calcium Phosphate Transfection Kit: Invitrogen Cat.No.K2780-01 Falcon tube (Cat No.35-2054:12 x 75 mm, 5 ml tube) Cell: 293
More informationG protein-coupled Signal Transduction
Theresa Filtz, hd har 735, Winter 2006 G protein-coupled Signal Transduction Main Objectives (the big chunks) Describe in molecular detail the cascades of events in a generalized G protein-coupled signaling
More informationReceptors and Drug Action. Dr. Subasini Pharmacology Department Ishik University, Erbil
Receptors and Drug Action Dr. Subasini Pharmacology Department Ishik University, Erbil Receptors and Drug Action Receptor Receptor is defined as a macromolecule or binding site located on the surface or
More informationChapter 20. Cell - Cell Signaling: Hormones and Receptors. Three general types of extracellular signaling. endocrine signaling. paracrine signaling
Chapter 20 Cell - Cell Signaling: Hormones and Receptors Three general types of extracellular signaling endocrine signaling paracrine signaling autocrine signaling Endocrine Signaling - signaling molecules
More informationLecture Outline. Hormones & Chemical Signaling. Communication Basics: Overview. Communication Basics: Methods. Four methods of cell communication
Lecture Outline Hormones & Chemical Signaling Communication Basics Communication Overview Communication Methods Signal pathways Regulation (modulation) of signal pathways Homeostasis... again Endocrine
More informationSmall Airways Disease. Respiratory Function In Small Airways And Asthma. Pathophysiologic Changes in the Small Airways of Asthma Patients
Small Airways Disease Respiratory Function In Small Airways And Relevant Questions On Small Airway Involvement In How can small airway disease be defined? What is the link between small airway abnormalities
More informationAsthma Management for the Athlete
Asthma Management for the Athlete Khanh Lai, MD Assistant Professor Division of Pediatric Pulmonary and Sleep Medicine University of Utah School of Medicine 2 nd Annual Sports Medicine Symposium: The Pediatric
More informationProtocol for Gene Transfection & Western Blotting
The schedule and the manual of basic techniques for cell culture Advanced Protocol for Gene Transfection & Western Blotting Schedule Day 1 26/07/2008 Transfection Day 3 28/07/2008 Cell lysis Immunoprecipitation
More information2-Deoxyglucose Assay Kit (Colorimetric)
2-Deoxyglucose Assay Kit (Colorimetric) Catalog Number KA3753 100 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information...
More informationSupporting Information. A Two-In-One Fluorescent Sensor With Dual Channels to. Discriminate Zn 2+ and Cd 2+
Electronic Supplementary Material (ESI) for RS Advances Supporting Information A Two-In-One Fluorescent Sensor With Dual hannels to Discriminate Zn 2 and d 2 Li-Kun Zhang, a Guang-Fu Wu, a Ying Zhang,
More informationPharmacological Assessment of the In Vitro Functional Selectivity of Aclidinium Bromide at M 3 and M 2 Muscarinic Receptors in Human Tissue
Pulm Ther (2015) 1:103 107 DOI 10.1007/s41030-015-0003-x BRIEF REPORT Pharmacological Assessment of the In Vitro Functional Selectivity of Aclidinium Bromide at M 3 and M 2 Muscarinic Receptors in Human
More informationANATOMY & PHYSIOLOGY - CLUTCH CH. 6 - CELL COMMUNICATION.
!! www.clutchprep.com CONCEPT: CELL-TO-CELL CONNECTIONS AND SIGNALING Gap and Tight Junctions: Adjacent cells communicate and hold on to each other via junctions. Two important kinds: Gap Junctions are
More informationSupplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after
Supplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after photoconversion by using H2B-Dendra2. 4-5 PPs of H2B-Dendra2 BM chimeras were photoconverted and analyzed 7 days (upper panel)
More informationMechanisms of action of bronchial provocation testing
Mechanisms of action of bronchial provocation testing TSANZ / ANZSRS Masterclass: April 3rd, 2016 13:00 13:30 John D. Brannan PhD Scientific Director - Dept. Respiratory & Sleep Medicine John Hunter Hospital,
More informationAnnexin V-PE Apoptosis Detection Kit
Annexin V-PE Apoptosis Detection Kit Catalog Number KA0716 100 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information...
More informationUpdate on Pulmonary Diseases. Jeffrey Lessar, MD
Update on Pulmonary Diseases Jeffrey Lessar, MD 1 No disclosures to make No conflicts 2 Goals Update on key changes in Pulmonary Therapy 3 Spirometry Spirometry FEV1- forced expiratory Volume in 1 Sec
More informationVascular reactivity in sepsis and platelet dysfunction in septic shock
Vascular reactivity in sepsis and platelet dysfunction in septic shock Benjamin Reddi Discipline of Physiology School of Medical Science University of Adelaide Thesis submitted for the degree of Doctor
More informationIslet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot
Islet viability assay and Glucose Stimulated Insulin Secretion assay Islet cell viability was determined by colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay using CellTiter
More informationNEUROGENIC CONTROL OF CANINE BRONCHIAL SMOOTH MUSCLE: COMPARISON OF THE NORMAL AND EXPERIMENTALLY-INDUCED HYPERRESPONSIVE STATES
NEUROGENIC CONTROL OF CANINE BRONCHIAL SMOOTH MUSCLE: COMPARISON OF THE NORMAL AND EXPERIMENTALLY-INDUCED HYPERRESPONSIVE STATES By LUKE JEFFREY JANSSEN, B.Sc., M.Sc. A Thesis Submitted to the School of
More informationInternational Journal of Cell Cloning 6: (1988) Research Laboratory of Blood Physiology, Hunan Medical College, Changsha, Hunan, PRC
Original Paper International Journal of Cell Cloning 6:290-295 (1988) The Influence of Histamine at Various Concentrations on the Cell Cycle State of Hematopoietic Stem Cells (CF'U-s) I"i Shounan, XU You-Heng
More informationCell Signaling part 2
15 Cell Signaling part 2 Functions of Cell Surface Receptors Other cell surface receptors are directly linked to intracellular enzymes. The largest family of these is the receptor protein tyrosine kinases,
More informationSupporting Information
Supporting Information Muraski et al. 10.1073/pnas.0709135105 SI Text Generation of Transgenic Animals. Pim-WT and Pim-DN cdnas were subcloned NheI/SmaI from pegfp-c1 Pim-1 and pegfp-c1 Pim-DN plasmids
More informationB16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small cell lung cancer
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2017 Experimental Methods Cell culture B16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small
More informationOptimization of the GeneBLAzer H2 CRE-bla HEK 293T Cell Line
GeneBLAzer Validation Packet Version No.: 1Sep Page 1 of 5 Optimization of the GeneBLAzer H CRE-bla HEK 93T Cell Line GeneBLAzer H HEK 93T DA Cells GeneBLAzer H CRE-bla HEK 93T Cells Catalog Numbers K17
More informationAlphaScreen : A Straightforward and Powerful Alternative to ELISA. Martina Bielefeld-Sévigny Ph.D., R&D Director
AlphaScreen : A Straightforward and Powerful Alternative to ELISA Martina Bielefeld-Sévigny Ph.D., R&D Director Overview AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle
More informationCellular Messengers. Intracellular Communication
Cellular Messengers Intracellular Communication Most common cellular communication is done through extracellular chemical messengers: Ligands Specific in function 1. Paracrines Local messengers (neighboring
More informationData Sheet TIGIT / NFAT Reporter - Jurkat Cell Line Catalog #60538
Data Sheet TIGIT / NFAT Reporter - Jurkat Cell Line Catalog #60538 Background: TIGIT is a co-inhibitory receptor that is highly expressed in Natural Killer (NK) cells, activated CD4+, CD8+ and regulatory
More informationReactivity of the isolated perfused rat tail vascular bed
Brazilian Journal of Medical and Biological Research (1997) 30: 891-895 Perfused rat tail vascular bed ISSN 0100-879X 891 Reactivity of the isolated perfused rat tail vascular bed A.S. França, L.V. Rossoni,
More informationPotent and Selective CRTh2 Antagonists are Efficacious in Models of Asthma, Allergic Rhinitis and Atopic Dermatitis
Potent and Selective CRTh2 Antagonists are Efficacious in Models of Asthma, Allergic Rhinitis and Atopic Dermatitis Laura L. Carter, Yoshi Shiraishi, Yooseob Shin, Laurence Burgess, Christine Eberhardt,
More informationImproved Stability of the LANCE Ultra Signal in Kinase Assays
Improved Stability of the LANCE Ultra Signal in Kinase Assays LANCE Ultra is a high throughput screening (HTS) technology platform optimized for homogeneous time-resolved fluorescence resonance energy
More informationInstructions for Use. APO-AB Annexin V-Biotin Apoptosis Detection Kit 100 tests
3URGXFW,QIRUPDWLRQ Sigma TACS Annexin V Apoptosis Detection Kits Instructions for Use APO-AB Annexin V-Biotin Apoptosis Detection Kit 100 tests For Research Use Only. Not for use in diagnostic procedures.
More informationCell Lysis Buffer. Catalog number: AR0103
Cell Lysis Buffer Catalog number: AR0103 Boster s Cell Lysis Buffer is a ready-to-use Western blot related reagent solution used for efficient extraction of total soluble protein in nondenatured state
More information