Chronic use of inhaled beta-agonists by asthmatics is associated with a loss of bronchoprotective effect and deterioration of asthma control. Beta-agonist-promoted desensitization of airway smooth muscle beta-2-adrenergic receptors, mediated by G protein-coupled receptor kinases and arrestins, is presumed to underlie these effects, but such a mechanism has never been demonstrated. Using in vitro, ex vivo, and in vivo murine models, we demonstrate that beta-arrestin-2 gene ablation augments beta-agonist-mediated airway smooth muscle relaxation, while augmenting beta-agonist-stimulated cyclic adenosine monophosphate production. In cultures of human airway smooth muscle, small interfering RNA-mediated knockdown of arrestins also augments beta-agonist-stimulated cyclic adenosine monophosphate production. Interestingly, signaling and function mediated by m2/m3 muscarinic acetylcholine receptors or prostaglandin E2 receptors were not affected by either beta-arrestin-2 knockout or arrestin knockdown. Thus, arrestins are selective regulators of beta-2-adrenergic receptor signaling and function in airway smooth muscle. These results and our previous findings, which demonstrate a role for arrestins in the development of allergic inflammation in the lung, identify arrestins as potentially important therapeutic targets for obstructive airway diseases.—Deshpande, D. A., Theriot, B. S., Penn, R. B., Walker, J. K. L. β-Arrestins specifically constrain β2-adrenergic receptor signaling and function in airway smooth muscle.

Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca2+]i release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein–coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca2+]i response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca2+]i response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca2+]i response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin–mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.

Bitter taste receptors (TAS2Rs) of the tongue likely evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants, leading to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased ASM [Ca2+]i in a Gβγ, PLCβ and IP3-receptor dependent manner which would be expected (like acetylcholine) to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM, and dilation of airways that was 3-fold greater than β-agonists. Relaxation by TAS2Rs is from a localized [Ca2+]i response at the cell membrane which opens BKCa channels leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in an asthma mouse model. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

Purpose

A study was performed to assess dosimetric characteristics of volumetric modulated arcs (RapidArc, RA) and fixed field intensity modulated therapy (IMRT) for Whole Abdomen Radiotherapy (WAR) after ovarian cancer.

Methods and Materials

Plans for IMRT and RA were optimised for 5 patients prescribing 25 Gy to the whole abdomen (PTV_WAR) and 45 Gy to the pelvis and pelvic nodes (PTV_Pelvis) with Simultaneous Integrated Boost (SIB) technique. Plans were investigated for 6 MV (RA6, IMRT6) and 15 MV (RA15, IMRT15) photons. Objectives were: for both PTVs V90% > 95%, for PTV_Pelvis: Dmax < 105%; for organs at risk, maximal sparing was required. The MU and delivery time measured treatment efficiency. Pre-treatment Quality assurance was scored with Gamma Agreement Index (GAI) with 3% and 3 mm thresholds.

Results

IMRT and RapidArc resulted comparable for target coverage. For PTV_WAR, V90% was 99.8 ± 0.2% and 93.4 ± 7.3% for IMRT6 and IMRT15, and 98.4 ± 1.7 and 98.6 ± 0.9% for RA6 and RA15. Target coverage resulted improved for PTV_Pelvis. Dose homogeneity resulted slightly improved by RA (Uniformity was defined as U5-95% = D5%-D95%/Dmean). U5-95% for PTV_WAR was 0.34 ± 0.05 and 0.32 ± 0.06 (IMRT6 and IMRT15), 0.30 ± 0.03 and 0.26 ± 0.04 (RA6 and RA15); for PTV_Pelvis, it resulted equal to 0.1 for all techniques. For organs at risk, small differences were observed between the techniques. MU resulted 3130 ± 221 (IMRT6), 2841 ± 318 (IMRT15), 538 ± 29 (RA6), 635 ± 139 (RA15); the average measured treatment time was 18.0 ± 0.8 and 17.4 ± 2.2 minutes (IMRT6 and IMRT15) and 4.8 ± 0.2 (RA6 and RA15). GAIIMRT6 = 97.3 ± 2.6%, GAIIMRT15 = 94.4 ± 2.1%, GAIRA6 = 98.7 ± 1.0% and GAIRA15 = 95.7 ± 3.7%.

Conclusion

RapidArc showed to be a solution to WAR treatments offering good dosimetric features with significant logistic improvements compared to IMRT.

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed Gs-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gβγ sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via β-agonist stimulation of the beta-2-adrenergic receptor (β2AR). Conversely, neither 5′-(N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the β-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to Gs-coupled receptors in ASM, GRK2/3 selectively attenuates β2AR signaling, yet relief of GRK2/3-dependent β2AR desensitization does not influence at least one important physiological function of the receptor.

Glucocorticoids (GCs) and protein kinase A (PKA)–activating agents (β-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma—excessive ASM growth—are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1β and TNF-α mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2–dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1β stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1β, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein–coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of β-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.

A Helical Tomotherapy™ (HT) Hi-Art II (TomoTherapy, Inc., Madison, WI, USA) has been one of the important innovations to help deliver IMRT with image guidance. On-board, mega voltage computed tomography (MVCT) detectors are used for imaging and dosimetric purpose. The two objectives of this study are: (i) To estimate the dosimetric and general capability (TomoImage registration, reconstruction, contrast and spatial resolution, artifacts-free image and dose in TomoImage) of on-board MVCT detectors. (ii) To measure the dosimetric parameters (output and energy) following major repair. The MVCT detectors also estimated the rotational output constancy well. During this study, dosimetric tests were repeated after replacing MVCT detectors and the target. fixed-gantry/fixed-couch measurements were measured daily to investigate; the system stability. Thermoluminescense dosimeter (TLD) was used during both the measurements subsequently. The MVCT image quality with old and new detectors was comparable and hence acceptable clinically. The spatial resolution was optimal and the dose during TomoImage was 2 cGy (well within the manufacturer tolerance of 4 cGy). The results of lateral beam profiles showed an excellent agreement between the two normalized plots. The output from the rotational procedure revealed 99.7% while the energy was consistent over a period of twelve months. The Hi-Art II system has maintained its calibration to within +/− 2% and energy to within +/− 1.5% over the initial twelve-month period. Based on the periodic measurements for rotational output and consistency in the lateral beam profile shape, the on-board detector proved to be a viable dosimetric quality assurance tool for IMRT with Tomotherapy. Tomotherapy was stable from the dosimetric point of view during the twelve-month period.

A Helical Tomotherapy (HT) Hi-Art II machine, Hi ART (TomoTherapy, Inc., Madison, WI, USA) was installed at our center in July 2007, and was the first machine in India. Image-guided HT is a new modality for delivering intensity modulated radiotherapy (IMRT). Dosimetric tests done include (a) primary beam alignment (b) secondary beam alignment (c) water tank measurements (profiles and depth doses) (d) dose rate measurements (e) IMRT verification, and (f) Mega voltage Computed Tomography (MVCT) dose. Primary and secondary beam alignment revealed an acceptable linear accelerator (linac) alignment in both X and Y axes. In addition, it was observed that the beam was aligned in the same plane as gantry and the jaws were not twisted with respect to gantry. The rotational beam stability was acceptable. Multi-leaf collimators (MLC) were found to be stable and properly aligned with the radiation plane. The jaw alignment during gantry rotation was satisfactory. Transverse and longitudinal profiles were in good agreement with the “Gold” standard. During IMRT verification, the variation between the measured and calculated dose for a particular plan at the central and off-axis was found to be within 2% and 1mm in position, respectively. The dose delivered during the TomoImage scan was found to be 2.57 cGy. The Helical Tomotherapy system is mechanically stable and found to be acceptable for clinical treatment. It is recommended that the output of the machine should be measured on a daily basis to monitor the fluctuations in output.

Dynamic multileaf collimator (DMLC) and static multileaf collimator (SMLC), along with three-dimensional treatment planning system (3-D TPS), open the possibility of tissue compensation. A method using electronic tissue compensator (ETC) has been implemented in Eclipse 3-D TPS (V 7.3, Varian Medical Systems, Palo Alto, USA) at our center. The ETC was tested for head and neck conformal radiotherapy planning. The purpose of this study was to verify the feasibility of DMLC and SMLC in head and neck field irradiation for delivering homogeneous dose in the midplane at a pre-defined depth. In addition, emphasis was given to the dosimetric aspects in commissioning ETC in Eclipse. A Head and Neck Phantom (The Phantom Laboratory, USA) was used for the dosimetric verification. Planning was carried out for both DMLC and SMLC ETC plans. The dose calculated at central axis by eclipse with DMLC and SMLC was noted. This was compared with the doses measured on machine with ion chamber and thermoluminescence dosimetry (TLD). The calculated isodose curves and profiles were compared with the measured ones. The dose profiles along the two major axes from Eclipse were also compared with the profiles obtained from Amorphous Silicon (AS500) Electronic portal imaging device (EPID) on Clinac 6 EX machine. In uniform dose regions, measured dose values agreed with the calculated doses within 3%. Agreement between calculated and measured isodoses in the dose gradient zone was within 3 mm. The isodose curves and the profiles were found to be in good agreement with the measured curves and profiles. The measured and the calculated dose profiles along the two major axes were flat for both DMLC and SMLC. The dosimetric verification of ETC for both the linacs demonstrated the feasibility and the accuracy of the ETC treatment modality for achieving uniform dose distributions. Therefore, ETC can be used as a tool in head and neck treatment planning optimization for improved dose uniformity.

A new generation telecobalt unit, Theratron Equinox-80, (MDS Nordion, Canada) has been evaluated. It is equipped with a single 60-degree motorized wedge (MW), four universal wedges (UW) for 15°, 30°, 45° and 60°. MW was configured in Eclipse (Varian, Palo Alto, USA) 3D treatment planning system (TPS). The profiles and central axis depth doses (CADD) were measured with radiation field analyzer blue water phantom for MW. These profiles and CADD for MW were compared with UW in a homogeneous phantom generated in Eclipse for various field sizes. The absolute dose was measured for a field size of 10 × 10 cm2 only in a MEDTEC water phantom at 10 cm depth with a 0.13 cc thimble ion chamber (Scanditronix Wellhofer, Uppsala, Sweden) and a NE electrometer (Nuclear Enterprises, UK). Measured dose with ion chamber was compared with the TPS predicted dose. MW angle was verified on the Equinox for four angles (15°, 30°, 45° and 60°). The variation in measured and calculated dose at 10 cm depth was within 2%. The measured and the calculated wedge angles were in well agreement within 2°. The motorized wedges were successfully configured in Eclipse for four wedge angles.