AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity  Autotaxin (ATX) and its lipid product lysophosphatidic acid (LPA) are key players in cell proliferation and motility and are thus central to both normal physiology and the physiology of several diseases. ATX is a mammalian extracellular lipase with lyosphospholipase D (lysoPLD) activity that hydrolyzes lysophosphatidylcholine (LPC) to the lysolipid mediator LPA and choline . LPA binENPP2) is among the 40 most upregulated genes in highly metastatic cancers Overexpression of ATX contributes to the pathophysiology of invasive cancers in vivo applications in vivo, both during drug development and during ATX-directed therapy. Such a method could (i) visualize ATX activity in preclinical animal models, (ii) identify patients that would benefit from ATX therapy, and (iii) once these patients are enrolled, could serve as a companion diagnostic. Knowing that a candidate ATX inhibitor is active against the target at both a molecular and a tissue level provides critical preclinical and potentially clinical information. Similar personalized medicine approaches are currently used in the clinic for other treatments Considerable effort is being devoted to develop drugs targeting the ATX/LPA axis with several promising drug candidates such as PF-8380, showing good bioavailability for ex vivo measurements of ATX or LPA levels in biological fluids. However, LPA turnover in serum is both rapid and exquisitely sensitive to sample acquisition, thereby limiting the utility of this approach Currently, monitoring the ATX/LPA pathway in animals relies on in vivo we developed a near-infrared imaging probe, AR-2  was appended to a mimic of the choline head group. IRDye® 800CW has optimal excitation and emission maxima for in vivo optical imaging  with many imaging platforms tuned to these fluorescence properties, and as a result, has been widely used for basic and translational research programs in vitroAR-2 was designed as an analog of LPC, in which a fluor (IRDye® 800CW) was linked to the in vitro by monitoring fluorescence with increasing amounts of purified ATX  hydrogel in vivo imaging for the MDA-MB-435 tumors will be reported elsewhere). Once xenotransplanted tumors were established, AR-2 was delivered intravenously, and NIRF was subsequently imaged in whole bodies of live animals. NIRF signals were tumor-specific and elevated compared to the rest of the body (e.g. in circulation) and the fluorescent product selectively accumulates in the tumor. Nevertheless, there is an increase in NIRF signal for ATX-expressing tumors that correlates with intra-tumoral ATX levels.To determine whether ATX can activate AR-2 the body , and in the body , MDA-MB-the body . These din situ. To determine the optimal timing for imaging, we investigated the pharmacokinetic properties of AR-2 in mice. The time course of AR-2 in the plasma after intravenous dosing shows bi-exponential decrease over time  of AR-2 is 432 mL/kg, b.w. (or 8.6 mL in 20 g mouse) close to 43% of body weight suggesting that AR-2 distributes extensively When intravenously administered, AR-2 produces a rapid rise of intense fluorescence signal in the whole body. The apparent volume of distribution  or with vehicle alone 20 min prior to AR-2 injection. High NIRF signals were clearly visible in tumors from vehicle-treated mice, with markedly lower signals observed in the PF-8380-treated group . To compin vivo “smart probe” capable of measuring and imaging ATX activity in living organisms. The AR-2 probe is a valuable tool with several potential clinical applications, including (i) discovery and development of pharmacological inhibitors targeting ATX, (ii) identification of ATX-expressing cancers and other ATX-related diseases, (iii) selection of patients who are candidates for ATX inhibitor treatment, (iv) development of a companion diagnostic for ATX-directed therapies, and (v) guiding surgical resection of ATX-expressing tumors. In addition, AR-2 will allow discovery of additional physiological and pathophysiological roles for ATX in vivo.Here, we describe the first Purified human ATX, INPP4A, LPLA2, PF-8380, S32826, BrP-LPA and FS-3 were obtained from Echelon Biosciences, Inc. . Mouse ATX, human NPP1, and mouse and human NPP7 were purchased from R&D Systems . Shrimp alkaline phosphatase was purchased from Promega . COS-7, MDA-MB-231, and MDA-MB-435 cell lines were purchased from ATCC . Extracel was purchased from the Glycosan division of BioTime, Inc . IRDye® 800CW and IRDye® QC-1 NHS esters were from LI-COR . Bradford reagent was purchased from Sigma-Aldrich .AR-2 was synthesized following the steps described for FS-3 substituting the NHS esters of the near infrared dye IRDye® 800CW and quencher IRDye® QC-1 2, 1 mM MgCl2, 140 mM NaCl, and 1 mg/mL fatty acid free bovine serum albumin. Reactions were immediately placed in a Spectramax M2 plate reader  set at 37°C, monitoring fluorescence exciting at 750 nm and emitting at 805 nm over time. Unless otherwise stated, all reactions used 10 nM enzyme, except alkaline phosphatase, which was performed at 0.1 unit/µL.AR-2 (100 nM) was incubated with the indicated enzyme in buffer containing 50 mM Tris-Cl pH 8.0, 5 mM KCl, 1 mM CaCl50 of each compound was calculated by fitting a standard curve using a sigmoidal dose-response (variable slope) nonlinear regression curve using GraphPad Prism (version 5.03).To test ATX inhibition, stock solutions of PF-8380, S32826 or BrP-LPA were prepared in DMSO then diluted with water to achieve the final experimental concentrations. DMSO was spiked into each reaction mixture to equalize vehicle concentrations. Purified human ATX  was mixed with varying concentrations of inhibitor at 37°C for 10 min, then AR-2 or FS-3 was added to a final concentration of 1 µM. The rates of fluorescence increase were measured for up to 2 h. The initial velocity of fluorescence increase was determined and normalized to control reactions. The ICm) was determined for each compound by measuring the rate of fluorescence increase at varying substrate concentrations using Prism to fit to the Michaelis-Menton equation. Inner filter effects were overcome using previously described methods i) for each set of reactions was determined from the IC50 following standard methods.The Michaelis constant (KCOS-7 cells grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and penicillin/streptomycin were transfected with human β ATX cDNA inserted into pcDNA DEST40  or mock plasmid in DMEM without FBS using TransIT®-2020 transfection reagent  according to the manufacturer's protocol.g for 1 min to collect cells. Conditioned media was prepared by concentrating the supernatant ∼40-fold using Amicon Ultra YM30 .MDA-MB-231 and MDA-MB-435 cells were grown to 80% confluence in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS. Cells were rinsed twice with PBS and grown for approximately 60 h in RPMI supplemented with 1% FBS. Media were centrifuged at 1000× Equal volumes of cell media (COS-7) or conditioned media (MDA-MB-231 or MDA-MB 435) were mixed with buffer to achieve a final concentration of AR-2 (1 µM). All other conditions were as described for purified enzymes.7 cells/mL and each mouse received 0.1 mL of Extracel tumor suspension. To reduce auto-fluorescence arising from the feed, mice were switched to specially formulated diet  at least two days prior to imaging.Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah (protocol numbers 10-10016 and 12-05013). Tumors in mice were established as previously described b.w. bolus. The animals were then imaged 48 h after injection with a Pearl Impulse Imager  using the 800 nm channel. The fluorescence signals from acquired images were analyzed using Pearl Impulse Software v 2.0 (LI-COR Biosciences) according the manufacturer's protocols. For ATX inhibition studies, PF-8380 was suspended in a hydroxyethyl cellulose vehicle at 2 g/L. Mice were given PF-8380  or vehicle orally 20 min prior to AR-2 injection. ATX-Red dosing, delivery and imaging were unchanged.Six weeks after tumor graft, mice were imaged using AR-2. To visualize tumors, AR-2 was diluted in PBS, filtered through a 0.2 µm porous membrane, and intravenously injected via the lateral vein at 0.5 mg/kg ex vivo fluorescence was imaged and quantified as previously described. Tumors were immediately frozen until further use. Thawed tumors were homogenized in ice cold buffer containing 10 mM Tris pH 7.4, 200 mM NaCl, 10% glycerin, 5 mM EDTA and 1× Halt protease inhibitor cocktail  at a ratio of 40 mg tumor to 1 mL buffer. Samples were centrifuged at 14,000× g at 4°C. ATX levels were determined by diluting supernatants ten times in RD5-10 and assayed using a Human ENPP-2/Autotaxin ELISA  and an Autotaxin Sandwich ELISA  using the manufacturers' protocol. Total protein levels were determined by the method of Bradford Animals were sacrificed, organs were dissected and weighed, then b.w. The animals were then lightly anesthetized with isoflurane for blood collection at 0, 0.08, 0.25, 0.5, 1, 2, 4, 6, 24, and 48 h after AR-2 administration. Blood samples were collected in EDTA coated tubes by submandibular bleeding or caudal vena cava (at time of sacrifice) with the maximum number of two blood collections from each animal. The collected blood samples were centrifuged, and the plasma was stored at −20°C until analysis.To investigate preliminary PK of AR-2, female BALB/c mice aged 8–10 wks old  were used. The animals were allowed to acclimatize to housing at least one week prior to use. At day 0, AR-2 was administered as a single bolus injection through lateral tail vein at a dosage of 1.0 mg/kg tert-butanol∶water (7∶1) then centrifuged at 14,000× g to pellet precipitated proteins. The resulting supernatant was frozen and lyophilized. Samples were then reconstituted in cold PBS and subjected to HPLC analysis using a C18 column  with a gradient of 5–60% acetonitrile in 50 mM triethylammonium acetate (pH 6.0). Peaks with absorbance at 778 nm were integrated and compared to mouse plasma samples containing known concentrations of AR-2. The resulting AR-2 concentration versus time data were plotted and fitted with a sum of two exponentials to calculate plasma half-life (t1/2) and various pharmacokinetic parameters using the PK package in R Statistics (version 2.15.1).To determine the concentration of AR-2 in plasma, samples were prepared according to the following procedures. Plasma samples were mixed with nine volumes of Figure S1Pharmacokinetics of AR-2. The time course of AR-2 in the plasma was fit to a bi-exponential equation yielding half-lives of 0.3 h and 8.5 h, respectively.(TIF)Click here for additional data file.Figure S2AR-2 Synthesis. All reagents were purchased from Aldrich or Acros and used without further purification. The NHS-esters of IRDye® QC-1 and IRDye® 800CW were purchased from Li-COR Biosciences. Chromatography was performed on an Isco Combiflash Companion using pre-packed C18 silica columns (Teledyne-Isco). 1H (400 MHz) and 31P (162 MHz) NMR spectra were recorded at 25°C on a Varian INOVA instrument. Chemical Shifts are given in ppm. Mass spectra were measured at the University of Utah Medicinal Chemistry Department using electrospray ionization (ESI). HPLC was performed on a Waters 2795 system with a 2990 diode array detector. The reverse phase column  was eluted with a linear gradient of 0–40% acetonitrile in 50 mM triethylammonium bicarbonate (pH 6) over 15 min. 2. Triethylamine (400 µL) was added to a solution of 124  and IRDye® QC-1 NHS ester  in dry DMF (12 mL) and the reaction was stirred for 2–3 hours protected from light. The solvents were evaporated under reduced pressure and the residue was co-evaporated twice with toluene. The product was purified by reverse phase chromatography using a linear gradient of, 5–40% acetonitrile in 50 mM triethylammonium acetate (pH 6.0). The desired fractions were pooled and dried under vacuum. Yield: 47.2 mg . 1H NMR (CD3OD): 8.40 , 8.25 , 8.13 , 8.05 , 7.89 , 7.82 , 7.37 , 7.13 , 6.83 , 6.67 , 5.84 , 4.45 , 4.14 , 3.88–4.00 , 3.63–3.82 , 3.41–3.63 , 3.38 , 3.30 , 3.20 , 2.84–2.94 , 2.78 , 2.68 , 2.61 , 2.35 , 2.15–2.26 , 2.12 , 1.92–2.04 , 1.84–1.90 , 1.82 , 1.723 , 1.69 , 1.44–1.61 , 1.27–1.43 , 0.78 , 0.01 , 0.00 . 31P NMR (CD3OD): 1.29. ESI-MS: 924.6 (M-2H)2−, 616.4 (M-3H)3−, 462.1 (M-4H)4−. AR-2. Trifluoroacetic acid (6 mL) was added to a solution of 2  in CH2Cl2 (20 mL) and stirred for 1 hour in the dark. The solvents were evaporated under reduced pressure then co-evaporated with toluene (2×10 mL),1∶1 MeOH∶toluene (10 mL). The residue was dissolved in dry DMF (6 mL) then IRDye®800CW-NHS ester  was added followed by followed by triethylamine (250 µL). The reaction was stirred in the dark for 2–3 hours then the solvent was evaporated under reduced pressure. The product was purified by reverse phase chromatography using a linear gradient of, 5–40% acetonitrile in 50 mM triethylammonium acetate (pH 6.0). The desired fractions were pooled and dried under vacuum. Yield: 32 mg . 1H NMR (CD3OD): 8.49 , 8.35 , 8.23 , 8.15 , 7.76–8.07 , 7.48 , 7.36 , 7.30 , 7.23 , 7.19 , 6.93 , 6.77 , 6.08 , 6.20 , 5.94 , 4.55 , 4.02–4.20 , 3.82–4.00 , 3.50–3.72 , 3.47 , 3.40 , 3.43 , 2.86–3.04 , 2.68–2.82 , 2.45 , 2.25–2.36 , 1.10–2.26 . 31P NMR (CD3OD): 1.64. ESI-MS: 654.8 (M-4H)4−, 523.6 (M-5H)5−, 436.5 (M-6H)6−, 373.7 (M-7H)7−. HPLC 11.03 min (95% at 778 nm).(TIF)Click here for additional data file.


Background. We aimed to apply the ZHENG identification to provide an easy and useful tool to stratify the patients with allergic rhinitis (AR) through exploring the correlation between the quantified scores of AR symptoms and the TCM ZHENGs. Methods. A total of 114 AR patients were enrolled in this observational study. All participants received the examinations of anterior rhinoscopy and acoustic rhinometry. Their blood samples were collected for measurement of total serum immunoglobulin E (IgE), blood eosinophil count (Eos), and serum eosinophil cationic protein (ECP). They also received two questionnaire to assess the severity scores of AR symptoms and quantified TCM ZHENG scores. Multiple linear regression analysis was used to determine explanatory factors for the score of AR manifestations. Results. IgE and ECP level, duration of AR, the 2 derived TCMZHENG scores of “Yin-Xu − Yang-Xu”, and “Qi-Xu + Blood-Xu” were 5 explanatory variables to predict the severity scores of AR symptoms. The patients who had higher scores of “Yin-Xu − Yang-Xu” or “Qi-Xu + Blood-Xu” tended to manifest as “sneezer and runner” or “blockers,” respectively. Conclusions. The TCM ZHENG scores correlated with the severity scores of AR symptoms and provided an easy and useful tool to stratify the AR patients. ZHENG diagnosis and therefore are prescribed different TCM herbal treatment.Allergic rhinitis (AR) is a common disease with a prevalence of at least 10% to 20% in western nations –5. Some ZHENG (syndrome), a basic unit and key concept in TCM theory, is the diagnosis made or concluded after careful analysis of all symptoms and signs. This process to achieve the diagnosis is based on the physiology and pathology of TCM, and is named “differentiation of ZHENG” or “ZHENG identification” [ZHENG identification, the treatment modalities are individualized and mainly based on the ZHENG which each patient is diagnosed with.ication” . This chication” . Guided  ZHENG is diagnosed according to each patient's clinical manifestations, it reflects more subtle individual differences in patients with the same disease, which may be a tool to predict the severity of AR symptoms or categorize the AR patients. The purpose of this study was to probe the correlation between the severity of AR symptoms and the TCM ZHENG, and also try to adopt the TCM ZHENG identification to provide an easy and useful tool to stratify the AR patients.Currently, there are no agreed ways to predict the severity of allergic rhinitis manifestations. Either nasal airway assessments, laboratory parameters, or physical findings usually show a low degree of correlation or a nonsignificant correlation with patients' symptoms –17. SincCandida, Alternaria, Aspergillus, Cladosporium, and Penicillium). A written informed consent was obtained from each participant. Exclusion criteria for this trial were those who (1) took antihistamine, decongestant, or had used topical steroids within 2 weeks, (2) were under 12 years old, (3) were pregnant, (4) were ongoing immunotherapy, or (5) were with severe physical or mental illness. The study protocols were approved by the Institutional Review Board of the Taichung Veterans General Hospital (IRB TCVGH no. 930116/279).This was an explorative, cross-sectional, and observational clinical trial. Patients diagnosed with AR were enrolled from the outpatient of the Otorhinolaryngology Department of Taichung Veterans General Hospital from 2004 to 2005. The diagnosis of AR was made by the specialist physician according to the clinical manifestations, history, and positive skin testing of a local screening panel  performed the anterior rhinoscopy for all eligible patients and graded their rhinoscopic findings. The edematous degree of inferior turbinate and the amount of nasal discharge were graded from 0 (none) to 3 (severe). Nasal minimal cross-sectional area (MCA) was assessed by acoustic rhinometry in each patient. The blood samples were collected from the patients for analysis of the inflammatory marker, including: total serum immunoglobulin E (IgE), blood eosinophil count (Eos), and serum eosinophil cationic protein (ECP).Each enrolled patient completed a self-report questionnaire assessment to assess the severity of allergic symptoms within the latest 2 weeks: this questionnaire, which also included questions on age, gender, family history, comobility , and duration of AR, was specific to the severity of allergic symptoms including nasal obstruction, sneezing, rhinorrhea, itchy nose, and itchy eye. These symptoms were graded from 0 to 3 according to the severity within previous 2 weeks .ZHENG Identification.” The diagnosis of TCM ZHENG is the summary of a specific functional state of the human body [ ZHENGs in TCM, either simple ZHENG or combined ones [In TCM, a disease is a common product of both pathogenetic factors and maladjustments in the body. The body must have the capacity to regulate itself in order to maintain homeostasis and adapt to the environmental stimulus. If the body's regulation ability fails to maintain homeostasis, then diseases may develop . Therefoman body . There aned ones .ZHENGs: “Yin-Xu, Yang-Xu, Qi-Xu, and Blood-Xu” were chosen to be measured in the AR patients. It was because Internal Classis, an important TCM literature, points out that: “Qi, Blood, Yin, and Yang” are 4 basic important elements to maintain the body's normal function; the physiological equilibrium and the circulation of Qi and Blood all change in response to the environmental variations. These responses help preserve the dynamic equilibrium of the body's Yin and Yang [ZHENGs: “Yin-Xu, Yang-Xu, Qi-Xu, and Blood-Xu” will be diagnosed . The measurement of this questionnaire provided a quantified and comparable parameter to explore the correlation between TCM ZHENGs and the severity score of AR symptoms. An easy-applied and standardized TCM ZHENG diagnosis instrument was developed by our research team through 2 rounds of TCM experts' meetings. After several discussions, according to the TCM theory and considering its use in western medical setting, this instrument was designed to measure the 4 basic TCM ZHENGs by answering the 24 questions which described the signs and symptoms of the 4 ZHENGs. These signs and symptoms in the previous 2 weeks were self-reported and assessed by a 4-point frequency and intensity scale (each was graded from 0 to 3). With higher scores of frequency and intensity, a more pronounced pathological status of each TCM ZHENG was indicated (the TCM ZHENG Questionnaire and the TCM ZHENG measured by each question are listed in the appendix). The Cronbach α coefficients of this questionnaire were 0.70, 0.64, 0.77, and 0.76 for the four TCM ZHENG domains of “Yin-Xu, Yang-Xu, Qi-Xu, and Blood-Xu,” respectively. The scores of “Yin-Xu, Yang-Xu, Qi-Xu, and Blood-Xu” represented the decreased level of Yin, Yang, Qi, and Blood to maintain normal function of the body  “Yin-Xu − Yang-Xu”: this derived from the score of Yang-Xu ZHENG subtracted from the score of Yin-Xu ZHENG, and denoted the TCM ZHENG “Yin-deficiency with Yang-hyperactivity.” Physiologically, the normal body function is maintained upon the harmonious cooperation and mutual restrain of Yin and Yang [Yin is unable to restrain Yang, some signs and symptoms of Yang-hyperactivity will manifest, since the level of Yin and Yang is expected to be equal in normal circumstance. So, the derived parameter score of “Yin-Xu − Yang-Xu” was aimed to quantify this pathological status  “nd Blood .ZHENG scores with the severity scores of AR symptoms. Multiple linear regression analysis with the total symptom score as the dependent variable was carried out to determine the variables independently associated with the severity of AR. Binary logistic regression was used to link the symptoms which impacted daily life (symptom score = 2 or 3) and TCM ZHENG score. Two-tailed P value <0.05 was considered statistically significant.Statistical analyses were conducted using the Statistical Package for the Social Science  version 12.0. The demographic characteristics of the observed patients were described by frequency, percentage, mean, and standard deviation. The correlation among the study variables was examined by Spearmen's rank correlation. A test for linearity was used to evaluate the trend of the TCM A total of 114 AR patients were enrolled in our study. Both the questionnaire assessment and anterior rhinoscopy were done for all the 114 patients; examination of acoustic rhinometry for 111, measurements of IgE for 109, measurement of ECP for 106, and measurement of Eos for 100. The basic characteristics of the patients are listed in There was no significant correlation between the AR patients' age and the total or each AR symptom score; neither the correlation between the smoking habit and the symptom score. Patients who had other allergic diseases  or family members  with allergic diseases  did not have higher symptom scores either. However, the duration of AR showed significant correlation with both the total symptom scores and the score of itchy eye .There was no correlation noted between the scores of rhinoscopic findings and the AR symptom scores. The sum of second nasal minimum cross-sectional area (MCA2) measured by acoustic rhinometry did not correlate with the symptom scores either .r = 0.206 and P = 0.034) and moderate correlation with sneezing (r = 0.164 and P = 0.093). The IgE level had moderate correlation with sneezing (r = 0.157 and P = 0.102)  .Yin-Xu” ZHENG correlated significantly with the total and each AR symptom score. The score of “Yang-Xu” ZHENG did not correlate with any symptom score. The scores of “Qi-Xu” and “Blood-Xu” ZHENG correlated significantly with the total symptom scores, the scores of nasal obstruction, and itchy nose and eye. The score of “Yin-Xu − Yang-Xu” correlated significantly with the total and each AR symptom score, except nasal obstruction. Another derivative parameter “Qi-Xu + Blood-Xu” correlated significantly with the total symptoms scores, the scores of nasal obstruction, and itchy nose and eye  factors for the severity scores of AR symptoms. Beside the correlated variables noted in the above bivariate correlation analysis, since age and sex were important demographic factors, they were put into the multiple linear regression model. Simultaneously, IgE was also added considering it being checked regularly in the clinical practice for AR patients. ZHENG scores were used as explanatory variables for regression analysis. The collinearity was solved by using the score of the derivative parameter “Yin-Xu − Yang-Xu” and “Qi-Xu + Blood-Xu” instead of the 4 basic TCM ZHENGs for the regression model.In the beginning of the multiple linear regression analysis, we faced the problem of collinearity when the 4 basic TCM P = 0.039), ECP level (P = 0.017), duration of AR (P = 0.016), the scores of “Yin-Xu − Yang-Xu” (P = 0.004), and the score of “Qi-Xu + Blood-Xu” (P = 0.015)  the original symptom scores graded from 0 to 1 were recategorized into “0,” which meant no impact on daily life; (2) the original scores graded from 2 to 3 were recategorized into “1,” which meant the symptoms had impact on daily life.Qi-Xu + Blood-Xu” . The symptoms of rhinorrhea and itchy nose which impacted daily life were correlated with higher scores of “Yin-Xu − Yang-Xu” . The symptom of itchy eye which impacted daily life (the score of itchy eye = 2 or 3) was correlated with higher scores of “Yin-Xu − Yang-Xu”  and longer duration of AR   .This explorative, cross-sectional, and observational clinical study adopted and integrated both the diagnostic method of western medicine and TCM in patients with AR. AR is defined as a symptomatic disorder of the nose induced after allergen exposure by an IgE-mediated inflammation. The update treatment guidelines initiated by the World Health Organization recommend classification of allergic rhinitis into “intermittent” (IAR) or “persistent” (PER) allergic rhinitis, instead of previous classification of “seasonal” or “perennial” allergic rhinitis , 2. It iZHENG based on the TCM theory after inquiry and physical examination. However, the theory of TCM is complicated and not easily realized by western medicine (WM) physicians and investigators. WM doctors are often skeptical about the validity of TCM clinical diagnosis [ZHENG Questionnaire” as a simplified mathematic model of TCM inquiry and provided an easy-applied, standardized diagnosis tool. Questions in the questionnaire were designed to survey patients' physical conditions based on the four basic TCM ZHENGs: “Yin-Xu, Yang-Xu, Qi-Xu, and Blood-Xu.” Higher scores in the questionnaire meant more pronounced pathological statuses. The TCM physical findings of patients' pulse, tongue, nails, lips, and face were not included in this study because of the difficulty in standardization.TCM doctors diagnose the iagnosis , 21. We ZHENG scores to be associated with the severity of AR by analysis of bivariate correlation. The inflammatory parameters correlated with the total symptom scores after adjusting the TCM ZHENG scores, meaning the TCM ZHENG scores were a confounding factor to the inflammatory parameters. Investigations of the predictors of the severity of allergic rhinitis manifestations are few and conflicting. Several studies have proved the relationship between IgE, ECP and Eos, and atopic diseases –25. HoweAcoustic rhinometry is a geographic measurement of the nasal cavity by using reflections of sound wave. The acoustic rhinometry is safe and its validity has been proven by comparison with measures obtained by computerized tomography or magnetic resonance imaging scanning –31. HoweZHENG (“Yin-Xu − Yang-Xu” and “Qi-Xu + Blood-Xu”), duration of AR, IgE, and ECP level were found to be good predictors for the severity scores of AR in our study. However, the R2 value was only 0.28, indicating some other factors could contribute to the severity of allergic rhinitis. We believe that environmental factors, or mucociliary function may play a role causing the severity of AR manifestations. The TCM ZHENG scores: “blockers”  having significant higher scores of “Qi-Xu + Blood-Xu”, while “sneezer and runner”  having higher scores of “Yin-Xu − Yang-Xu”. From the TCM pathological point of views, these findings were very reasonable and closely fitted to the TCM theory. Since the nasal obstruction may be caused by the deficiency of Qi and/or Blood; while the rhinorrhea or itchy nose or eye are the manifestations of Yin-deficiency with Yang-hyperactivity. These results showed that the TCM ZHENG diagnosis correlated with the modern western medicine, and the TCM ZHENG diagnosis could reflect subtle differences among the patients with AR. Khanna and Shah  reportedTo our knowledge, this was the first study which adopted the TCM diagnostic questionnaires to quantify disease-specific severity and to categorize the patients with AR. Our results revealed that the TCM diagnostic questionnaires can be used similarly to disease specific quality of life standardized questionnaires such as the SF-36  or the RQLQ (a disease-specific QOL) instrument. Our research team had conducted several clinical studies which adopted both the TCM diagnostic questionnaires and the quality of life standardized questionnaires. It was found that the results measured by the 2 questionnaires were comparable in several aspects. Our team will report these interesting findings and new application of TCM diagnosis continuously.ZHENG score, the duration of AR, and the IgE and ECP level were found to be independently and significantly explanatory of the severity of AR manifestations. The TCM ZHENG diagnosis correlated with the modern western diagnosis and may provide a novel approach to stratify the AR patients. These findings may provide a new applied field of TCM ZHENG diagnosis.The TCM


BOLD (blood oxygen level dependent) fMRI  is commonly used to study differences in neuronal activity between human populations. As the BOLD response is an indirect measure of neuronal activity, meaningful interpretation of differences in BOLD responses between groups relies upon a stable relationship existing between neuronal activity and the BOLD response across these groups. However, this relationship can be altered by changes in neurovascular coupling or energy consumption, which would lead to problems in identifying differences in neuronal activity. In this review, we focus on fMRI studies of people with autism, and comparisons that are made of their BOLD responses with those of control groups. We examine neurophysiological differences in autism that may alter neurovascular coupling or energy use, discuss recent studies that have used fMRI to identify differences between participants with autism and control participants, and explore experimental approaches that could help attribute between-group differences in BOLD signals to either neuronal or neurovascular factors. Because fMRI is a powerful and non-invasive tool to examine brain function in humans, it is often employed to read out neuronal activity in such studies. However, for fMRI-based measures of brain activity to be reliably compared between populations, it must be assumed that the relationship between neuronal activity and the BOLD signal is the same in the different groups. This relationship depends on the mechanism by which active neurons alter local blood flow (neurovascular coupling) and on the oxygen use evoked by neuronal activity .Several neurophysiological changes have been identified in autism (as discussed below). These changes may alter the signalling from neurons to the vasculature and, in consequence, the relationship between neuronal activity and the BOLD signal. Typically, studies using fMRI to compare control participants and participants with autism attribute observed differences in the BOLD signal to altered neuronal activity, without considering the possibility that neurovascular coupling or oxygen consumption is also altered . More reIn this article, we start by briefly reviewing the physiological basis of the BOLD signal and the neurovascular coupling mechanisms that mediate it. We then look in detail at the neurophysiological changes that are known to occur in autism and how these changes might influence neurovascular coupling and oxygen use, and thus the BOLD response. We then examine the implications that altered neurovascular coupling or oxygen use would have for fMRI studies. By focusing on several representative studies chosen from the literature, we highlight the interpretational problems that can occur when this issue is ignored. Finally, we explore ways in which the scientific community studying autism can address the challenge of separating out neurovascular coupling and neuronal activity effects on the BOLD signal experimentally, as some groups are already beginning to do.2fMRI is regularly used to study neuronal activity in humans because of its non-invasive nature, which allows it to be used to study a broad range of participants, including patients suffering from pathologies such as autism. It is important to note, however, that the signal produced – the BOLD signal – is not a direct measure of neuronal activity, but instead reflects the operation of several different brain processes associated with neuronal activity, including synaptic transmitter release, the resulting release of signalling molecules that mediate neurovascular coupling and thus increase local blood flow, and  oxygen consumption .The BOLD signal reports changes in the amount of deoxyhaemoglobin in the blood. Deoxyhaemoglobin is paramagnetic, and it decreases MRI signals by making the local magnetic field less homogenous. During neuronal activity, the level of deoxyhaemoglobin initially increases as oxygen use increases, and then decreases as a blood flow increase occurs which over-compensates for the amount of oxygen used. This decrease in the level of deoxyhaemoglobin results in a more homogeneous magnetic field and thus increases the magnetic resonance signal. The size of the BOLD signal is larger when there is a large blood flow increase, but is decreased by the use of oxygen by neurons . The effect of decreased inhibition on neuronal activity can be detected by fMRI: because GABA decreases excitability, an autistic brain with impaired GABA function would experience more neuronal excitability and therefore more neuronal firing and more glutamate release, which triggers the release of vasodilators and increases blood flow  is impaired in autism. Compared to controls, a reduction in the expression of ETC genes  and prot7The medication used to treat autism may also affect the relationship between neuronal activity and the BOLD response. For example, serotonin reuptake inhibitors (SSRIs), a medication commonly used by autistic patients, increase extracellular serotonin levels in the brain. They may, therefore, increase the tone of vessels – perhaps beyond normal levels – and thus alter the blood flow response evoked by neuronal activity (see above). A recent study by 8Having highlighted some of the neurophysiological differences found in autism that may alter the relationship between neuronal activity and the BOLD response in this condition, we will now consider some fMRI studies in which the results may be affected by these differences.9Since most researchers are interested in the neuronal changes associated with autism, most changes in fMRI response are simply assumed to be due to altered neuronal processing rather than altered neurovascular coupling or oxygen use. None of the studies above considered neurovascular abnormalities in autism, which, if present could directly affect the BOLD signal even in the absence of changes in neuronal activity. The mislabelling of neurovascular changes as neuronal activity changes could be misleading. The best way to avoid this is with an experimental design that can distinguish a neurovascular coupling (or oxygen use) change from a neuronal activity change. A few labs have begun to design experiments with this goal in mind.Since Feczko et al. found no significant difference in the visual task-evoked BOLD response between ASD and control children, they argued that neurovascular coupling in the two groups must be the same, ideally throughout the brain. This is the most parsimonious argument, but the authors do acknowledge the small possibility that a confounding difference in neurovascular coupling that has equal and opposite effects to a difference in neuronal activity between the groups could lead to the same overall BOLD signal. For example, a smaller amount of neuronal activity in autism may be compensated for by a stronger neurovascular signalling pathway, or vice versa. In this case, a similar blood flow response would be produced in the different groups by differing levels of neuronal activity.Even if one accepts the argument that a similar BOLD response for a low level visual task implies similar neurovascular coupling, as noted by the authors, it can only be made for this particular visual task and need not generalise to other tasks. As mentioned above, this is because different tasks activate different brain regions, different neuronal pathways and different neurotransmitter systems, which vary in their neurovascular coupling mechanisms . Indeed,Such input- or task-specific neurovascular coupling makes it difficult for results based on one task alone to be extrapolated usefully to general claims about neurovascular coupling between groups – it is always a possibility that the chosen task happens to be one in which neuronal responses and neurovascular coupling are not altered between groups, while for tasks employing different brain regions and pathways there may indeed be differences of neurovascular coupling between the groups. For example, in Feczko et al.’s case, it is possible that low level visual areas do not have their neurovascular coupling altered by the neuronal changes associated with autism, while brain areas dealing with social interactions do have their neurovascular coupling altered. Nevertheless, this study represents the first attempt to experimentally rule out neurovascular coupling confounds in the interpretation of fMRI data, and paves the way for paradigms which address this issue directly.This line of reasoning on its own is weak, because there is no reason why a neurovascular coupling change could not selectively alter task-evoked signalling to the vasculature without affecting resting state blood flow. This is obvious when one considers that the majority of neurovascular coupling changes are likely to occur at points along the pathway from synaptic transmission to vessel dilation/constriction, and may therefore only be revealed during task-evoked synaptic activity.Nevertheless, Dinstein et al.’s conclusions are supported by EEG data showing that visual task-evoked response variability is significantly greater in participants with ASD . The com10Autism is a brain disorder, with its diverse behavioural phenotypes rooted in abnormalities of neuronal circuitry and function, and we do not wish to suggest that fMRI cannot reveal these neuronal differences. The aim of this review is to dispel the idea that neurovascular coupling can be lumped in with easily corrected for non-neuronal confounds such as head motion or breathing artefacts. On the contrary, neurovascular coupling changes can have profound, complex and region- and task-specific effects on cerebral blood flow and therefore the BOLD signal.Autism is associated with many neurophysiological changes, and there are just as many reasons why an abnormal relationship between neuronal activity and the BOLD signal might therefore be expected. Being aware of these potential neurovascular coupling and energy use differences will be critical to the field's ability to dissect out the neuronal changes that underpin behavioural differences. Eventually, detailed knowledge of the physiology of the BOLD signal  will allThe authors declare no conflict of interest.


Multivariate neural data provide the basis for assessing interactions in brain networks. Among myriad connectivity measures, Granger causality (GC) has proven to be statistically intuitive, easy to implement, and generate meaningful results. Although its application to functional MRI (fMRI) data is increasing, several factors have been identified that appear to hinder its neural interpretability: (a) latency differences in hemodynamic response function (HRF) across different brain regions, (b) low-sampling rates, and (c) noise. Recognizing that in basic and clinical neuroscience, it is often the change of a dependent variable  between experimental conditions and between normal and pathology that is of interest, we address the question of whether there exist systematic relationships between GC at the fMRI level and that at the neural level. Simulated neural signals were convolved with a canonical HRF, down-sampled, and noise-added to generate simulated fMRI data. As the coupling parameters in the model were varied, fMRI GC and neural GC were calculated, and their relationship examined. Three main results were found: (1) GC following HRF convolution is a monotonically increasing function of neural GC; (2) this monotonicity can be reliably detected as a positive correlation when realistic fMRI temporal resolution and noise level were used; and (3) although the detectability of monotonicity declined due to the presence of HRF latency differences, substantial recovery of detectability occurred after correcting for latency differences. These results suggest that Granger causality is a viable technique for analyzing fMRI data when the questions are appropriately formulated. Granger causality Neuronal mechanisms of cognitive operations are inferred by comparing dependent variables across experimental conditions. For example, in attention research, neuronal responses under attend versus non-attend conditions are compared, whereas in working memory research, neuronal responses under different levels of working memory load are compared. It has been suggested that rather than focusing on the magnitude of Granger causality under a single experimental condition, it is more informative to focus on how it is modulated by experimental conditions We generated simulated neuronal data using a bivariate autoregressive (AR) model with model order = 1:The noise covariance matrix The neural time series were convolved with a canonical HRF Consider two simultaneously recorded stationary time series They can also be jointly represented as an unrestricted bivariate AR model:In Eq. (4), Then For practical data analysis the above infinite sums need to be truncated to sums with finite orders. For simulated neural data, the AR model order was determined to be 1 according to the Bayesian Information Criterion (BIC) In each experiment, fMRI Granger causality (GC) was plotted against the corresponding neural GC, and the relation was assessed by Spearman rank correlation. If the correlation between fMRI GC and neural GC along the same direction  is significantly positive then it was taken as evidence for monotonicity. In contrast, if fMRI GC in one direction  was significantly correlated with the neural GC in the opposite direction (X→Y), the monotonicity was considered false, caused by HRF convolution, downsampling, and noise addition. Previous work has shown that in unidirectional coupling, if X→Y is nonzero but Y→X is zero at the neuronal level, HRF convolution, downsampling, and noise can lead to nonzero Y→X at the fMRI level, which is spurious  were the number of experiments in which neural GC and fMRI GC along the same directions were significantly positively correlated, and To quantify the simulation results, true positive rate (TPR) and false positive rate (FPR) of monotonicity detection were estimated, depending on whether fMRI GC and neural GC along the same direction and along opposite directions were significantly positively correlated. Specifically, in the unidirectional case, we defined TPR, FPR, and TDR were plotted as functions of different fMRI TRs, varying from 50 ms to 4 s, to study the impact of sampling rates on detecting the monotonicity relationship. Similar analysis was carried out by varying the noise level between 5% (SNR = 20) and 160% (SNR = 0.625).In addition to low sampling rates and noise, another factor impacting fMRI GC analysis is HRF latency variability. We considered this issue in the case of bidirectional coupling. Across 100 experiments the HRF latency difference between X and Y followed a normal distribution with zero mean and standard deviation (SD). Varying SD from 0.2 s to 1 s, we plotted TPR, FPR, and TDR as functions of SD under slow sampling (TR = 2 s) and fast sampling (TR = 50 ms), respectively. To mitigate the adverse effects of HRF latency variability, we followed a procedure given by Chang et al. In the bivariate AR(1) model in Eq. (1), the coupling strength for X→Y was between c = 0 and c = 0.8, and the coupling strength for Y→X was set at zero (b = 0). Convolving neural data with HRF yielded HRF-convolved neural signal which, after downsampling to realistic TRs  and addition of white noise , became fMRI time series. This process is illustrated in For TR = 2 s and 20% noise (SNR = 5), a positive correlation between fMRI X→Y GC and neural X→Y GC was clearly seen , indicatTo examine the effects of sampling rates, we plotted TPR, FPR and TDR as functions of different TRs. The noise level was fixed at 20% (SNR = 5). Although TPR decreased as TR increased, seen in To examine the effects of noise, we plotted TPR, FPR and TDR as functions of different noise levels, with TR fixed at TR = 2 s. As seen in Two HRFs with different peak times are shown in By applying the latency correction technique (see Method), we found that the distributions of correlation coefficient are better separated as shown in Further, we plotted TPR, FPR, and TDR, before and after latency correction, as functions of the standard deviation of the HRF latency difference distribution, under long and short fMRI TR  has emerged as a useful technique to evaluate directional influences in multivariate electrophysiological data. For fMRI data, however, its validity is debated. Previous simulation work has shown that convolution with HRF, low sampling rate, and addition of noise can yield spurious Granger causality According to the linear transform model, fMRI signal is closely related to locally averaged neural activity such as local field potentials (LFPs) via HRF The goal of our work is different from some of the recent works on the applicability of Granger causality to fMRI data. Rather than focusing on detecting network configuration  Detecting network configuration relies on statistical significance testing to ascertain whether a given GC is larger than zero or not. Past simulations have shown that such testing cannot prevent the detection of spurious Granger causality at the fMRI level, and that noise and downsampling can create nonzero GC when there is none at the neural level For the case of unidirectional coupling , the neural GC and the corresponding HRF-convolved GC for X→Y had nearly identical values, and the monotonicity relationship was clearly held with a slope close to 1 . In addiUsing TPR, FPR and TDR to characterize the results, we found that although TPR decreased when more stringent correlation significance threshold was employed, the FPR was always significantly lower than TPR and approached zero faster . This meThe numerical results above are further supported by analytical and numerical results presented in Low sampling rate is a major reason for deteriorated Granger causality estimation at the fMRI level The adverse effects of noise on Granger causality estimation have been studied in the past Different brain regions may have different hemodynamic response profiles 2 inhalation method and the breath holding (BH) method A possible remedy for HRF latency induced deterioration of GC estimation is to estimate HRF latency and correct for it. We show that if HRF latency can be determined experimentally, then with latency correction In the current simulation study we only considered pairwise GC. In the real brain the causal interactions between two brain areas may be mediated by a third brain area. Identifying and accounting for this “third variable” is therefore important in figuring out how information is routed in brain circuits. Conditional Granger causality is one way to deal with this problem Real world neural time series are far more complex than that generated by AR (1). The assumed steps in transforming neural time series to fMRI time series, including HRF convolution, down-sampling and noise addition, are only an approximation of the actual physiological cardio-neural coupling In a recent paper we considered the behavioral consequences of the interaction between the dorsal attention network (DAN) and the ventral attention network (VAN) In another recent paper, applying the same approach, we investigated the interactions between the so-called task control network (TCN) and the default mode network (DMN). It has been hypothesized that TCN exerts top-down control by issuing signals to regulate activities in different brain areas to facilitate task performance Appendix S1Mathematical analysis of effect of downsampling and HRF convolution on Granger causality. This Appendix has two objectives. First, we provide an analytic treatment of the effect of downsampling on Granger causality for an autoregressive (AR) model of order 1 (AR(1)), to further demonstrate that Granger causality from the downsampled signal is an monotonic function of Granger causality from the original signal. Second, we show that HRF convolution preserves and in fact enhances the monotonic relationship.(DOCX)Click here for additional data file.


Pregnant rats were fed control (CON) or MLP diets (n = 12 each group) throughout pregnancy. When aged 6 months, hearts from offspring underwent Langendorff cannulation to assess contractile function during baseline perfusion, 30 min ischemia and 60 min reperfusion. CON male hearts demonstrated impaired recovery in left ventricular pressure (LVP) and dP/dtmax (P < 0.01) during reperfusion when compared to MLP male hearts. Maternal diet had no effect on female hearts to recover from IR. MLP males exhibited greater membrane expression of β2-AR following reperfusion and urinary excretion of noradrenaline and dopamine was lower in MLP and CON female rats versus CON males. In conclusion, the improved cardiac recovery in MLP male offspring following IR was attributed to greater membrane expression of β2-AR and reduced noradrenaline and dopamine levels. In contrast, females exhibiting both decreased membrane expression of β2-AR and catecholamine levels were protected from IR injury.A maternal low-protein diet (MLP) fed during pregnancy leads to hypertension in adult rat offspring. Hypertension is a major risk factor for ischaemic heart disease. This study examined the capacity of hearts from MLP-exposed offspring to recover from myocardial ischaemia-reperfusion (IR) and related this to cardiac expression of  Protein restriction had no effect on recovery of the female heart [Adult cardiovascular disease is associated with metabolic and physiological aberrations that occur during fetal development. Epidemiological studies provide evidence that intrauterine growth retardation increases the risk of hypertension and cardiovascular disease in adult life . Such file heart . This sele heart .β-adrenergic signalling is key to cardiac contractile function and cardiac recovery following IR. The catecholamines adrenaline and noradrenaline activate myocardial cell surface β-adrenergic receptors. β1 and β2-AR are the principal β-receptor subtypes in the heart making up approximately 70–80% and 30–20% of the β-ARs expressed, respectively [β1 and β2-AR subtypes whereas noradrenaline acts mainly through β1-AR [β-AR activation involves release of the stimulatory Gαs subunit from the receptor. This stimulates adenylyl cyclase activity and generation of cAMP- and PKA-mediated activation of the contraction-relaxation cycle [β2-AR activation which acts in an antagonistic fashion by coupling Gαi leading to the attenuation of cAMP-mediated activation of PKA. Continual β-AR activation results in phosphorylation and receptor desensitisation through G protein uncoupling [ectively . Adrenalh β1-AR . β-AR acon cycle , 14. Thecoupling . The recβ-AR signalling in the heart has been widely investigated and established that myocardial ischaemia is commonly associated with an increase in membrane expression of β-ARs and that their overexpression prior to ischaemia increases IR-injury [β-AR signalling in developmental programming are yet to be explored. This study therefore aims to examine the capacity of isolated adult rat hearts from protein-restricted offspring to recover from IR injury in relation to alterations in shuttling of β-AR and G protein signalling between the sarcolemma and cytosolic compartments of the myocardium.The interaction between IR injury and R-injury –19. Howen = 12) or a low-protein diet , as described previously in detail [Twenty-four virgin Wistar rat dams  weighing approximately 250 g were mated in the animal facilities at the University of Nottingham. The appearance of a semen plug on the cage floor confirmed that mating and rats were allocated either a control (CON) diet , anaesthetised using 3% isofluorane in 2 litres O2/min and killed by cervical dislocation. The heart was rapidly excised and cannulated via the aorta to Langendorff perfusion apparatus within 90 seconds  and perfused with Krebs Henseleit buffer ) bubbled with O2-CO2  in a coronary retrograde fashion. Perfusion pressure was maintained at a constant pressure of 60 mmHg, with perfusate warmed to 37.4°C, and the heart immersed in a water-jacketed temperature-controlled glass chamber set at 37.4°C, therefore ensuring normothermia throughout the perfusion protocol. Contractile function was monitored by the careful insertion of a saline filled latex balloon (Linton Instruments) into the left ventricle which was adjusted to an end diastolic pressure of 5–10 mmHg. Left ventricular and perfusion pressure were continuously monitored through precalibrated physiological pressure transducers . Data recording was not started until all variables were stable (15–30 min) after which the following 30 min were defined as baseline. Ischemia was then induced by switching off the coronary perfusion apparatus for a 30-minute period. Coronary perfusion was then reinstated for a further 60-minute period to assess cardiac responses during reperfusion. Data for left ventricular developed pressure (LVP), heart rate (HR) and left ventricular first derivative (dP/dtmax) were collected and processed using the Powerlab Data Acquisition System (AD) Instruments. To determine the effect of IR on myocardial expression of β-AR, hearts from CON or MLP offspring that underwent either (i) 30 min baseline perfusion alone (n = 9-10), (ii) 30 min baseline perfusion followed by 30 min ischemia (n = 9-10) or (iii) 30 min baseline perfusion followed by 30 min ischemia and 60 min reperfusion (n = 9-10) on the Langendorff apparatus were collected and snap frozen for western blot procedures.At 6 months of age, male and female rats from each CON and MLP litter were randomly selected .Hearts collected for western blot analysis from the Langendorff study were used to prepare cardiac membrane and cytosolic fractionated samples as described by Fernandez-Twinn and colleagues . Frozen β1-AR , β2-AR , G stimulatory protein (Santa Cruz Gαs (K-20): sc-823), and G inhibitory protein (Santa Cruz Gαi-3 (C10): sc262). Membranes were incubated in blocking solution (5% dried skimmed milk in TBS with 1% Tween 20) prior to incubation with primary antibodies. Horseradish peroxidise secondary antibody conjugated to rabbit IgG was used at a working concentration of 1 : 5000 . Bands were developed on high performance chemiluminescence film  using ECL Plus reagent . Densitometric analysis of band intensity across gels was performed using a Biorad Gel Doc XR imaging system and Quantity One 1D analysis software. Three replicates of a pooled extract were included on every gel and used as a standard to facilitate gel-to-gel comparisons.Protein concentrations of the supernatant and resuspended pellet fractions were determined using the Bio-Rad protein assay system  according to the manufacturer's instructions. Samples were standardised to a concentration of 3 mg/mL with Laemmli's sample buffer  and boiled for 3 min before equal protein quantities of each sample were separated by SDS PAGE. Proteins were transferred to nitrocellulose membrane  for probing with antibodies to Rats at 5 weeks, 10 weeks, and 6 months of age were housed in metabolic cages for 24 hours and urine collected as previously described . Urine cdP/dtmax relative to baseline were analysed using a one-way repeated measure ANOVA. Western blot data were analysed by three-way ANOVA considering maternal diet, sex, and ischemia-reperfusion treatment as fixed factors. ELISA data were analysed by three-way ANOVA considering maternal diet, sex, and age as fixed factors. Measurements from related animals from the same litter were grouped in all ANOVA analyses and assigned as a random factor. All statistical analyses were performed using SPSS version 18. In all analyses, P < 0.05 was considered statistically significant.All results are presented as mean values with their standard errors. All data were analysed for homogeneity of variance, and if heterogeneous transformed by square root or logarithmic conversion. For the analyses, ischemia-reperfusion data were split into three distinct time bins: (1) 30 min baseline, (2) recovery 1 (0–30 min of reperfusion), and (3) recovery 2 (31–60 min of reperfusion). The effects of maternal diet on the recovery in LVP, HR, and dP/dtmax baseline indices in either sex  to ischemia-reperfusion insult (P < 0.01)  and a trend (P = 0.06) for an improved recovery during the R2 phase in comparison to CON hearts  and second (R2) 30 min periods of reperfusion was measured in Langendorff-perfused hearts from male and female rats. Maternal diet had no effect on LVP, HR, or ther sex . Similarn insult . However < 0.01) . A similN hearts . In contN hearts .β adrenergic signalling is a central mechanism involved in the maintenance of cardiac inotropic and chronotropic function and has been shown to play a key role in recovery of the heart from IR. Accordingly, we quantified levels of the two main β-AR isoforms, β1-AR and β2-AR, as well as levels of the stimulatory and inhibitory G proteins Gαs and Gαi, respectively, in isolated hearts exposed either to baseline perfusion (B), baseline perfusion plus ischemia (I), or baseline perfusion plus ischemia plus reperfusion (IR). Expression was measured in both membrane (membrane  and cytomembrane  fractionβ1-AR levels at the membrane increased following ischemia and returned to baseline levels by the end of 60 min reperfusion (P < 0.05). This correlated with a decline in β1-AR in the cytosol by the end of the ischemic period which had declined further by the end of reperfusion (P < 0.001). A significant interaction between diet and sex (P < 0.05) was also observed for membrane expression of β1-AR indicating that the overall level of receptor expression in females was lower in the MLP group when compared to CON, an effect not observed in males.β2-AR revealed a significant interaction between maternal diet, sex, and ischemia-reperfusion treatment (P < 0.05). β2-AR decreased in CON females following ischemia which was in complete contrast to all other groups where receptor expression levels were unchanged from baseline levels. Following reperfusion, β2-AR expression in CON females remained unchanged from levels observed after ischemia. Hearts from CON males showed decreased membrane expression of β2-AR after reperfusion similar to the decreased expression observed in CON female hearts following ischemia (β2-AR expression throughout ischemia and reperfusion.Cardiac membrane expression of ischemia . Howeverβ2-AR expression in cardiac cytosolic fractions was found to increase after ischemia and increased further following reperfusion (P < 0.001) which was in direct contrast to β1-AR cytosolic expression. These findings suggest that ischemia-reperfusion initiates a differential response in membrane trafficking of β1-AR and β2-AR from the cytosol to the cardiac membrane. Despite changes in β-AR isoform within the cardiac membrane and cytosol, no overt changes were observed in the levels of Gs and Gi (s and Gi  proteinsP < 0.02). In addition, a significant effect of age was observed where noradrenaline concentrations were significantly higher at 5 and 10 weeks of age respectively (P < 0.02). An interaction between age and sex was also evident (P < 0.01) where urine concentrations of noradrenaline in females were highest at 5 weeks of age with a concentration of 15 ng/mL before dropping to 8 ng/mL at 10 weeks of age, and remaining at this level when aged 6 months. For males, noradrenaline concentration at 10 weeks of age had risen from 10 ng/mL observed at 5 weeks to 14 ng/mL before dropping by 60% to 8 ng/mL at 6 months of age.Concentrations of the catecholamines adrenaline, noradrenaline, and dopamine were measured in the urine of rats at 5 weeks, 10 weeks, and 6 months of age. No significant differences were observed in the concentration of adrenaline at each stage of development . Urine lP < 0.01), and an interaction between sex and age was also observed (P < 0.04). Female rats had a significantly reduced urinary excretion of dopamine at 6 months of age compared to both 5 and 10 weeks of age. In contrast, urinary excretion of dopamine in male rats increased significantly with age. At 6 months of dopamine concentrations were significantly higher than the level determined at 5 and 10 weeks of age.Dopamine concentrations were significantly reduced by an MLP diet (The key finding from the present study is that hearts from MLP male offspring demonstrated improved recovery in cardiac function following IR when compared to CON males, whereas IR recovery in females was unaffected by maternal diet. This study verifies that sex is an important factor in the capacity of the heart to recover from IR injury , 22–24. β-AR signaling, we determined protein expression of the β-AR isoforms, and the Gs and Gi proteins in baseline-perfused ischemia or ischemia-reperfused hearts. β1-AR expression was associated with increased expression within the plasma membrane after ischemia that returned to baseline levels following reperfusion. This suggests that the β1-AR expression is upregulated in the membrane in response to ischemia and declines throughout reperfusion. This agrees with Strasser et al. [β1-AR levels increase following 50 min ischemia that was associated with decreased adenylyl cyclase activity. More recently, β1-AR protein expression in isolated rat hearts has also been demonstrated to increase at the plasma membrane following 30 min ischemia but with no change in adenylyl cyclase activity [β1-AR density at the plasma membrane decreases following 60 min reperfusion compared to levels after ischemia which was associated with a doubling in adenylyl cyclase activity [β1-AR expression following ischemia and reperfusion in the present study may not be reflective of downstream changes at the level of adenylyl cyclase.To investigate how cardiac function during IR relates to r et al. , who ideactivity . Furtheractivity . It is iβ2-AR in the cardiac membrane decreased following ischaemia in CON females and following reperfusion in CON males and MLP females. These findings demonstrate subtle sex-specific responses in downregulation of β2-AR at the cardiac membrane to ischaemia and reperfusion. To our knowledge, this is the first time sex-specific effects of IR on β2-AR abundance have been reported. A previous study focussing solely on male hearts reported that β2-AR density at the plasma membrane was unaffected following 30 min ischemia but decreased with 60 min reperfusion, thus agreeing with the present findings in CON males [β2-AR protein expression in the cardiac membrane of MLP males did not change throughout ischemia and reperfusion from baseline levels. Consequently, β2-AR levels were higher in MLP males compared to all other treatment groups following reperfusion.Protein expression of ON males . In contβ-AR density rather than G protein content at the cardiac membrane is the rate limiting component mediating cardiac contractility. Alterations in expression of β-AR isoforms through transgenic mouse models show profound effects on haemodynamic function of the heart [β2 rather than the β1-AR activation in cardiac pathology due to its specific capacity to enhance Ca2+ homeostasis and protection against apoptosis [β2-AR of the heart [β2-AR overexpression led to progressive cardiac hypertrophy and delayed onset cardiomyopathy. Moreover, β2-AR overexpression exacerbated ischemic injury but in the hearts of male mice only [β2-AR were protected from damage following IR and linked to the female sex hormone oestrogen acting through endothelial nitric oxide synthase to prevent Ca2+ dysregulation in the tissue [β2-AR expression changes. This may explain why cardiac β-AR expression levels observed between CON and MLP groups in the present study resulted in differences in haemodynamic parameters in male rats only.All these changes in BAR expression at the membrane occurred against a background where neither Gs nor Gi expression levels changed, suggesting that he heart , 33–35. poptosis . Howeverhe heart , 34, 35.ice only . Female e tissue . Such fiβ2-AR overexpression and does not represent a moderate increase in β2-AR expression [β2-AR overexpression have shown that 100–360-fold increases in β2-AR expression resulted in the onset of cardiomyopathy, whereas a moderate 60-fold increase had no overt pathological consequence [β2-AR signalling against cardiac pathology [β2-AR agonist treatment in conjunction with a β1-AR blocker was found to improve survival and contractility in a rat model of dilated cardiomyopathy [β2-AR agonist clenbuterol alone was sufficient to exert a cardioprotective effect in rat hearts through a reduction in myocardial apoptosis and oxidative stress and improving diastolic function following IR injury [β2-AR activation is thought to occur via stimulation of Gi rather than Gs-mediated signalling mechanisms which enable Ca2+ regulation to be maintained and apoptosis to be inhibited.The cardiac pathology observed in transgenic mouse models is possibly an artefact of such high pression . Comparisequence . Indeed athology –39. Longmyopathy . FurtherR injury . This caβ2-AR protein expression within the cardiac membrane following IR, which is in complete contrast to CON males. We propose that downregulation of β2-AR in response to IR is the cause of impaired contractile recovery in CON male hearts. Cardiac membrane expression of β2-AR also decreased after reperfusion in CON and MLP female hearts to a level comparable to CON males but despite this decrease showed no impairment in contractile recovery following ischemia. This may result from the protective role of oestrogen in maintaining a favourable Ca2+ microenvironment within female hearts during reperfusion that limits tissue damage and counteracts any changes in β-adrenergic signalling activity that would otherwise impair contractile recovery after ischemia. Measurements of urinary catecholamine levels indicate that by 6 months of age there is no overt difference in either adrenaline or noradrenaline levels in these animals. However, we did observe an increase in dopamine levels in males compared to female rats at 6 months of age. It is not clear how this difference affects cardiac function of these hearts. Dopamine is routinely administered as a therapy in heart failure to improve cardiac function [β-ARs in the heart. We did not see any evidence to indicate that this sex-specific difference in dopamine had any impact on basal levels of expression of any of the β-adrenergic signalling components measured in this study or on baseline activity of the hearts prior to ischemic reperfusion. Further work is required to explore other potential receptor targets of dopamine which may alter cardiac function in response to ischemia reperfusion.Our key finding is that MLP male hearts do not downregulate function . This isβ-ARs, decreasing β1 and increasing the β2 subtype although total β-AR content remained the same [Urine excretion of noradrenaline and dopamine was significantly lower in MLP compared to CON animals and may offset the ischaemic damage observed in CON males. Evidence to support this is the fact that depletion of catecholamines by surgical denervation causes a redistribution of the same . Furtherthe same .β2-AR expression at the cardiac membrane. Likewise, protein expression of β2-AR at the cardiac membrane decreased in response to IR in female CON and MLP hearts. However they displayed an improved cardiac recovery compared to CON males suggesting specific protective mechanisms in female hearts that counteract alterations in β-adrenergic receptor expression. Our findings indicate that in male offspring prenatal protein restriction maintains cardiac membrane expression of β2-AR during IR which improves cardiac contractile recovery. In contrast, female hearts were resistant to these effects suggesting subtle sex differences in the molecular mechanisms controlling cardiac membrane expression of β-AR in response to IR.We propose that the contractile dysfunction in male CON hearts following IR occurs as a result of increased catecholamine production and decreased


In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß1-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß1-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß1-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß1-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß1-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß1-AR complex. Therefore, bipartite binding of SAP97 to the ß1-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß1-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß1-adrenergic receptosome involved in connecting the ß1-AR to trafficking and signaling networks.Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß Cy3-conjugated to anti FLAG M2 IgG was purchased from Sigma (St. Louis MO). Anti-SAP97 antibody from Stressgen , anti Pan PDZ antibody  anti-ß1-AR antibodies, for human (sc-567) or rodents (sc-568), were purchase from Santa Cruz Biotechnology , AKAP79 from BD, . The siRNA sequence to human SAP97, 5′-GATATCCAGGAACATAAAT-3′ or its control 5′-CCATAATACAAGGTATAA-3′ were cloned into two vectors, one was the pcDNA6.2-GW-miR vector and the other was the pcDNA6.2-GW/EmGFP-miR vector to generate shRNA and EGFP-shRNA constructs, respectively .The ß-isoform of rat SAP97 cDNA was mutagenized using the Quick-Change II XL kit (Agilent Technologies) to introduce unique restriction sites flanking each of the three PDZs of SAP97. These restriction enzymes were selected because they were not found in SAP97 and generate blunt-ended cDNAs that can be fused together to create the desired SAP97 PDZ deletion construct. HpaI and PmlI sites were introduced at positions 660 and 924 flanking PDZ1. An AfeI site was introduced at position 1200, which lies downstream of PDZ2. A BsrB1 and SnaB1 sites were introduced at positions 1380 and 1644 to flank PDZ3. The expected lengths of PDZ1-PDZ3 were 88, 92 and 88 amino acids, respectively. Finally, KpnI and ApaI sites were introduced at the 5′- and 3′-ends of SAP97 by PCR. This modified SAP97 construct was used to create the various PDZ deletion mutants. Individual SAP97 PDZs fused to GST were generated by PCR and cloned pGEX-4T-2. Cloning the SmaI-XhoI fragment of the human ß1-AR into pGEX-4T-2 generated a N-terminal GST fusion to the carboxy-tail of the human ß1-AR encoding amino acids 425–477. In addition the carboxy-tail of the ß1-AR with hSAP97 shRNA were incubated with Cy3-conjugated, anti-FLAG M2 IgG (4 µg/ml) for 1 h at 37°C. Cells were treated with 10 µM isoproterenol for 30 min at 37°C to promote agonist-mediated receptor internalization. Then the cells were chilled to stop endocytosis and exposed to 0.5 M NaCl, 0.2 M acetic acid (pH 3.5) for 4 min on ice to remove antibody bound to extracellular ß1-AR 1-AR, respectively. Pixels were plotted as a function of time after the removal of isoproterenol in order to calculate the recycling kinetics of the ß1-AR HEK-293 cells stably expressing the FLAG-tagged WT ßCells expressing the appropriate constructs were lysed in radioimmune precipitation (RIPA) buffer . Equal amounts of clarified lysates were added to ∼5 µl M2 anti-FLAG-agarose beads. Control experiments were performed by incubating lysates with pre-immune IgG at the same concentration for 4 h at 4°C. The immune complexes were washed three times in RIPA buffer, eluted and the eluates were subjected to Western blotting.1-AR. After 48 hrs, the myocytes were lysed in RIPA buffer with protease and phosphatase inhibitors. The clarified lysate was mixed with 12 µl of anti-HA affinity beads for 24 h at 4°C. The resin was eluted and equal amounts of eluate were subjected to Western blotting and probed with anti SAP97, anti ß1-AR antibody and anti-AKAP150 antibody.Rat hippocampal crude membrane fractions were prepared in the presence of protease inhibitors and washed by resuspension and re-centrifugation in RIPA buffer without detergents. Washed membranes were solubilized in complete RIPA buffer and the cleared lysates were used for immunoprecipitation as described above. Neonatal cardiac myocytes were isolated from newborn wild type FVB mouse pups and cultured in 60-mm dishes for 24 h av for 30 min at 4°C. GST or GST-ß1-AR(425–477) or MBP-fusion proteins were incubated with cell lysates along with 10 µl-of glutathione- or amylose-agarose beads for 2 h at 4°C. After washing three times with lysis buffer, the proteins were eluted from the beads with 2X-laemmli sample buffer containing 20 mM dithiothreitol. Eluates were separated on SDS polyacrylamide gels, electroblotted to nitrocellulose filters and analyzed for SAP97 by immunoblotting.For pull-down assays, GST-fusion proteins were purified from BL21 bacteria by affinity chromatography on glutathione- or amylose-conjugated resins and then dialyzed in PBS containing 10% glycerol. HEK-293 cells expressing the various SAP97 constructs were lysed with 0.2% Triton X-100 in PBS supplemented with protease inhibitors and then clarified by centrifugation at 16,000×g1-AR(425–477) fusion in hybridization buffer  for 14 h at 4°C 1-AR(425–477) was detected by the anti-ß1-AR antibody.Far-western assays were performed by spotting 10 µl or 20 µl of a 1 µg/ml solution of GST or GST-PDZ onto nitrocellulose filters. The filters were blocked for 1 h with 3% bovine serum albumin in TBST and then incubated with 16 µg/ml or ∼0.34 µM of purified MBP-ß1-AR were transfected with PDZ2 of SAP97 in pIRES-EGFP or with K323A/326A-PDZ2 in pIRES. The cells were surface-biotinylated with 1.5 mg/ml sulfo-NHS-SS-biotin (Pierce) in Hanks’ balanced salt solution with Ca2+ and Mg2+ at 4°C. Biotinylated cells were exposed to isoproterenol for 30 min and then cooled to 4°C to stop membrane trafficking, and the remaining surface biotin was quantitatively cleaved with glutathione. After cleavage, warm DMEM was added, and cells were incubated at 37°C for 15, 30, and 60 min to allow internalized receptor to recycle before the cells were cooled to 4°C and incubated with glutathione cleavage buffer for a second time to ensure complete cleavage of any newly appearing surface biotin. At the end of each time point, the cells were lysed and processed as described Cells expressing the WT ß1-AR and either the scrambled shRNA-green fluorescent protein (GFP) or hSAP97 shRNA-GFP were used to determine the effect of SAP97 on desensitization and resensitization of adenylyl cyclase activities. In addition, in cells stably expressing the WT ß1-AR, we determined the effect of transiently transfected PDZ2-in pIRES-EGFP or K323A/326A-PDZ2 in pIRES on desensitization and resensitization. In all these experiments, the cells were cultured on 15 cm plates that were divided into four sets. The first and second sets were used as control for desensitization and the third and fourth sets for resensitization assays. For desensitization, cells were exposed to 1 mM ascorbic acid (control) or 10 µM isoproterenol for 30 min at 37°C, and then processed for the preparation of membranes as described act ± S.E. for isoproterenol versus 5 µM forskolin was calculated.HEK 293 cells stably expressing the WT ßt-test was used to determine significance, while multiple groups were compared by one-way (ANOVA) with Newman-Keuls post-hoc tests using Prism software .All data are expressed as means ± SE, except where indicated. For comparison between two groups of data, Student’s unpaired 18 peptide (PDB ID: 2G2L) was used as a template for computational modeling 1-AR was built based on the backbone coordinates of the GluR-A15-18 using AMBER89 force field in built-in homology modeling suit of MOE software . The resulting peptide was checked by MOE’s Protein Geometry Stereo-chemical Quality Evaluation tools in order to confirm that the model’s structure is consistent with crystallographic data on similar sequences. ESKV was then substituted for GluR-A15–18 in template PDZ-peptide complex X-ray crystal structure of SAP9797 PDZ2 bound to the GluR-Aet al1-AR, but the affinity of this interaction was not determined. Therefore, we mixed increasing concentrations of purified GST-ß1-AR(425–477) or GST with lysates prepared from HEK-293 cells over-expressing SAP97-YFP  of MBP-ß1-AR c-tail fusions  in SAP97 that bind to the type-1-PDZ of the ßof SAP97 . To iden fusions . These er values . These FLAG immunoblots also revealed that the 65-kDa FLAG-ß1-AR was the major immunoreactive band in the input from ß1-AR expressing HEK-293 cells (lane 1). However, in order to detect specific PDZ-ß1-AR interactions in lanes 3–10, the input from HEK-293 cells in lane 2 was overexposed revealing some non-specific immunoreactive proteins.Next we expressed and purified the individual GST-SAP97 PDZs and demonstrated that these proteins migrated with expected Mr values . Then eq of 1 ml . GST pulml . Next, we determined the effect of knock down of SAP97 on trafficking of the WT ß1-AR (images a–n). Cells stably expressing the WT ß1-AR with the scrambled EGFP-shRNA (images a–g) or with the EGFP-SAP97 shRNA (images h–n) were incubated with Cy3-labeled anti-FLAG M2-IgG  for 1 h and then visualized by confocal microscopy. Expression of either EGFP-shRNA did not interfere with the membranous distribution of the ß1-AR (images a and h). Exposing these cells to isoproterenol resulted in rapid endocytosis of membranous ß1-AR into intracellular vesicles (images b and i). After the removal of isoproterenol, the cells were incubated in an acetic acid/NaCl solution that stripped the surface-exposed antibody (ISO+A/W). This procedure revealed that internal ß1-AR were distributed into discrete punctate vesicular structures (images c and j). The effect of each shRNA on recycling of internal ß1-AR was initiated by removing isoproterenol followed by the addition of the ß-adrenergic receptor antagonist alprenolol to block further internalization, so that the recycling of internal ß1-AR could be unambiguously determined. In cells expressing the scrambled EGFP-shRNA, the agonist-internalized ß1-AR recycled back to the cell membrane within 60 min from the removal of isoproterenol (images d–f). However, the ß1-AR did not recycle when SAP97 was knocked down (images k–m). In cells expressing the scrambled EGFP-shRNA the ß1-AR recycled with a t0.5 = 18±5 min . Acid wash of cells expressing the scrambled EGFP-shRNA stripped >80% of the Cy3 pixels indicating that recycled ß1-AR were externalized (image g). On the other hand, Cy3 pixel distribution in SAP97 knockdown cells was not altered by this maneuver, indicating that the ß1-AR remained inside the cells that expressed the hSAP97 EGFP-shRNA (image g to n).A major consequence of GPCR recycling is externalization, which involves the re-insertion of the internalized GPCR into the cell membrane rnalized image g.1-AR in SAP97 knock down cells, we used HEK cells stably expressing FLAG-tagged ß1-AR and untagged hSAP97 shRNA (images o-b’). Rat SAP97 is resistant to hSAP97 shRNA because of sequence mismatches in the shRNA-targeted region. These cells were transiently transfected with either the empty p-IRES-EGFPII vector (images o-u) or with rat SAP97 in pIRES-EGFP (images v-b’). pIRES-EGFP is a bicistronic vector that allows the expression of the gene of interest to be monitored at the single-cell level due to expression of hrGFP II (green) on the same transcript. Images of cells that were pre-incubated with Cy3-labeled anti-Flag M2 IgG (red) for 1 h revealed that expression of empty pIRES-EGFP or rSAP97 in pIRES-EGFP did not interfere with the membranous distribution of the ß1-AR . Exposing these cells to isoproterenol resulted in rapid endocytosis of membranous ß1-AR into intracellular vesicles . Expression of the empty pIRES vector did not rescue the recycling of the ß1-AR in SAP97 knock-down cells (images r–u), while expression of rat SAP97 rescued the recycling of the ß1-AR and promoted its externalization (images y-b’). Expression of rSAP97 in SAP97 knockdown cells rescued the recycling of the ß1-AR, which recycled with a t0.5 of 21±6 min (1-AR in scrambled shRNA expressing cells vs. its recycling in SAP97 shRNA/rSAP97 expressing cells were comparable (p>0.05). On the other hand the recycling kinetics of the ß1-AR in these two cell types vs. the cell line expressing the SAP97 shRNA were significantly different (p<0.01 or p<0.001), as determined one-way ANOVA with Newman-Keuls post-hoc tests.To determine if rat SAP97 could rescue the recycling of the WT ß21±6 min . Analysi1-AR externalization and resensitization was examined by means of desensitization/resensitization assays of adenylyl cyclase activity in membranes prepared from the cells described in 1-AR and the scrambled or the SAP97 shRNA as described in the Materials and Methods section . The effect of rescuing SAP97 expression on resensitization of adenylyl cyclase activity was determined by transient overexpression of rSAP97 in pIRES-EGFPII in a double stable cell line that expressed the FLAG-WT ß1-AR, and hSAP97 shRNA .The relationship between ß section . To deteed shRNA . On the 97 shRNA . Express1-AR and hSAP97 shRNA were used in these studies. Each SAP97 deletion construct in pIRES-EGFP was transiently transfected into the double stable cell line and the trafficking of the ß1-AR was measured in EGFP expressing cells. In rSAP97 constructs with a single PDZ deletion such as ΔPDZ1 or ΔPDZ3, we observed normal recycling of internal ß1-AR and these receptors were externalized into the cells membrane (images d–g and r–u). In cells expressing rSAP97 in which PDZ2 was deleted, internal ß1-AR failed to recycle and were retained intracellularly (images k–n). In cells expressing double PDZ deletions, we observed that the ß1-AR did not recycle when PDZ2 was deleted such as in ΔPDZ1,2 or ΔPDZ2,3 . However when PDZ2 was retained, such as in ΔPDZ1,3, the ß1-AR recycled normally and was externalized (images f’ to i’). These data indicate that PDZ2 was required for mediating the effect of SAP97 on recycling and reinsertion of the ß1-AR into the cell membrane.The effects of specific PDZ deletions in the full-length ß-isoform of SAP97 were generated in the pIRES-EGFP II vector . HEK-2931-AR in HEK-293 cells. Cells doubly stable for the expression of FLAG ß1-AR and SAP97 shRNA were transiently transfected with plasmids expressing either full-length rSAP97 in pIRES-EGFP or the rSAP97 mutants described in panel) Resin eluates were divided into two portions, where by, ∼10% of each of the eluates were subjected to Western blotting and probed with the anti-ß1-AR antibody (top panel). These data indicated that equal amounts of FLAG-tagged ß1-AR were immunoprecipitated from each cell line. The remainder of each eluate (∼80%) were subjected to Western blotting and probed with anti-SAP97 to index SAP97 co-IP (middle panel). The data indicated that the ß1-AR co-IP’d rSAP97 from rSAP97 constructs that contained PDZ2, such as SAP97ΔPDZ1, ΔPDZ3 and ΔPDZ1,3. However, SAP97 constructs in which PDZ2 was deleted, such as ΔPDZ2, ΔPDZ1,2 and ΔPDZ2,3 were not co-IP’d by the ß1-AR. Therefore, the recycling data and the co-IP results indicated that PDZ2 was required for the interaction between SAP97 and the ß1-AR, which was a priori for recycling of the ß1-AR to occur.Next we determined the interaction between the SAP97 constructs of 1-AR .The ß-isoform of SAP97 contains at its N-terminus ∼200 amino acids L27 and a U1 domains that are involved in homo- and heterodimerization 1-AR  A and B.1-AR et al1-AR (image h) or with its internalization by isoproterenol (images i–j). However, internal ß1-AR did not recycle in cells expressing ΔL27-rSAP97-I2I5 and were retained by >85% intracellularly (images k-n). On the other hand, internal ß1-AR recycled efficiently in cells expressing ΔL27-rSAP97-I3I5 (images d–g).SAP97 also interacts with AKAP79/150, which is an A-kinase anchoring protein that binds PKA, PKC and protein phosphatase 2B  al1-AR image h 1-AR, we transfected the following constructs into the FLAG-tagged WT ß1-AR and hSAP97 shRNA double-stable cell line. We used SAP97-ΔL27/U1 in pIRES or a double deletion construct in which L27 and I3 were deleted . However, FLAG IP’s from cells expressing ΔL27-SAP97-I2I5 did not co-IP AKAP79 (lane 3). These data reiterate the findings of Gardner et al1-AR was indirectly mediated by SAP97. Thus, deleting the I3 domain from SAP97 interfered with its binding to AKAP79 without affecting its binding to the ß1-AR. The data in 1-AR.Next, we tested the interaction of these constructs with AKAP79 . FLAG ß1P AKAP79 , lane 3.1-AR and SAP97. Extracts prepared from cells stably expressing FLAG ß1-AR and SAP97-YFP were mixed with 2.5 µg of GST or molar equivalent of GST-PDZ . The result of GST pull down in 1-AR was mediated by PDZ2. Finally, we determined if in vivo overexpression of PDZ2 would interfere in the binding between the ß1-AR and SAP97. Each individual PDZ of SAP97 in pIRES-EGFP was transfected into HEK cells stably expressing the FLAG ß1-AR and SAP97-YFP . However, FLAG IP’s from cells expressing PDZ2 did not co-IP SAP97, indicating that PDZ2 interfered in the binding between the ß1-AR and SAP97 (lane 3).To determine the affinity of PDZ2-binding to the ß protein . These eAP97-YFP . FLAG IPnd SAP97 , lane 3.1-AR recycling, K323A/K326A rSAP97-YFP was expressed in SAP97 knockdown HEK cells . However, the K323A/K326A SAP97 construct could not rescue the recycling of internalized ß1-AR and these receptors were retained by >80% intracellularly (images d–g).Physical interactions between PDZ domains and PDZ type-1 ligands mainly involve the binding of the terminal carboxylates of the hydrophobic residue at P = 0 in the PDZ type-1 ligand with the ßB domain of PDZ2, as well as binding between the serine/threonine at P = -2 with a highly conserved histidine in the alpha/B alpha-helix of PDZ2  and [25]EK cells . This co1-AR by transiently transfecting WT rSAP97-YFP or K323A/K326A rSAP97-YFP into cells stably expressing FLAG ß1-AR and hSAP97 shRNA. FLAG IP’s of cell extracts prepared from these cells, indicated that WT rSAP97-YFP was co-IP’d with the ß1-AR, but K323A/K326A rSAP97-YFP was not IP’d (lane 3 to lane 4). These data show that lysines 323 and 326 of SAP97 PDZ2 were involved in SAP97 binding to the ß1-AR. To strengthen this argument we modeled the binding between the ESKV sequence corresponding to the last four amino acids in the carboxy tail of the ß1-AR to the published coordinates for the crystal structure of PDZ2 in complex with the C-terminal peptide (ATGL) of GluR1 Next we determined whether the K323A/K326A mutant would bind to the ß1-AR. We hypothesized that if PDZ2 binds selectively to the ß1-AR, then its overexpression would destabilize the ß1-AR-SAP97 scaffold and interfere with trafficking of the ß1-AR. Expression of the empty pIRES-EGFP vector or its fusions with individual PDZ’s did not affect the membranous distribution of the ß1-AR  or its internalization in response to isoproterenol . Expression of isolated PDZ1 or PDZ3 did not interfere with the recycling of the WT ß1-AR or with its externalization (images k–n and y-b’). Expression of isolated PDZ2 however, prevented efficient recycling of internal ß1-AR (images r-t). Acid treatment of these cells did not strip the Cy3 pixels, indicating that the majority  of internal ß1-AR remained inside the cell (image u). These data show that PDZ2 interfered with the binding of the ß1-AR to SAP97 . The cells were then exposed to isoproterenol for 30 min, followed by cleavage of the remaining cell surface biotin (lanes 2 and 7). The amount of biotin recovered in this step indexed the amount of biotinylated ß1-AR that was internalized in response to isoproterenol. Thus, the ratio of internal biotin to total biotin incorporated into the ß1-AR indexed the percentile of surface ß1-AR that was internalized in response to isoproterenol. When compared to WT PDZ2, it appeared that K323A/K326A PDZ2 reduced the amount of internalized ß1-AR in HEK-293 cells by ∼40% . Internalized ß1-AR were allowed to recycle by warming the cells (at 37°C) for an additional 15, 30, or 60 min (lanes 3–5 and 8–10). After each time period, the cells were cooled to 4°C and cleaved for the second time to cleave newly appearing surface biotin. Thus, the loss of biotin from the second cleavage step indexed recycling of the ß1-AR. The data indicate that by 30 min more than 90% of the biotin was lost from the ß1-AR in cells expressing K323A/K326A PDZ2 of SAP97, reflecting membrane recycling of the ß1-AR and subsequent biotin cleavage (lanes 3–5). In contrast, internalized (biotinylated) ß1-AR in HEK cells expressing WT PDZ2 were not markedly changed even after 1 h from the removal of isoproterenol, reflecting their internal distribution (lanes 9 and 10 with lanes 4 and 5). The results of 1-AR and endogenous SAP97 via PDZ2 was required for recycling of the ß1-AR in HEK-293 cells.In this series of experiments we sought to determine the effect of each isolated PDZ of SAP97 on trafficking of the ßthe cell image u.to SAP97  and inteto SAP97 . AnotherZ2-pIRES . An adva1-AR indicated that mutagenesis of K323 and K326 to alanine abrogated the ability of PDZ2 to pull down the ß1-AR .Pull down assays between PDZ2-GST or K323A/326A-PDZ2-GST and the ßhe ß1-AR . The eff1-AR, we transfected cells stably expressing the WT ß1-AR with empty pIRES-EGFP II, or with PDZ2 in pIRES-EGFP or K323A/K326A PDZ2 in pIRES-EGFP . The activity of resensitized adenylyl cyclase in membranes prepared from cells expressing K323A/K326A PDZ2 was 56±6% of maximal activity, which was not significantly different from the 67±7% activity in membranes prepared from non-desensitized K323A/K326A PDZ2 expressing cells (n = 5). Comparisons between the activities of adenylyl cyclase in membranes prepared from PDZ2 resensitized cells versus their K323A/K326A PDZ2 counterpart indicated that the activity of adenylyl cyclase in K323A/K326A PDZ2 membranes was significantly higher . Therefore, mutagenesis of K323A/K326A in PDZ2 interfered with the dominant negative activity of PDZ2 on recycling and resensitization of the ß1-AR signaling pathway.To determine the effect of PDZ2 on resensitization of the ßRES-EGFP . AgonistRES-EGFP . Overexp1-AR was explored in cells derived from neonatal mouse ventricular myocytes that endogenously express the ß1-AR, SAP97 and AKAP150, which is the rodent homolog of AKAP79 1-AR then solubilized and the ß1-AR was immunoprecipitated from the clarified lysates with anti-HA agarose  in SAP97 that interacted with the ß1-AR PDZ. The methodical analysis identified the PDZ2 domain of SAP97 as the primary binding cassette to the PDZ ligand in the ß1-AR. This finding is significant because PDZ3 was previously reported as the PDZ that binds to the ß1-AR 1-AR adds one more major signaling molecule to an already impressive retinue of PDZ2 binding partners, that include GluR1 and GluR2, E6 polypeptide of human papillomavirus (HPV), the type-1 somatostatin receptor, potassium voltage channels and others 1-AR, “ESDV” in the NR2B subunit, “ETQV” in the HPV-18-E6 and “ATGL” in the GluR1 subunit . The 3D crystal structure of PDZ2 bound to the C-terminal polypeptides of GluR1 and HPV18-E6 polypeptide showed that despite the apparent difference in the sequences of their carboxy termini, the C-terminal hydrophobic amino acid at position (P = 0) and the highly conserved Ser/Thr amino acid at position (P = −2) of both sequences were bound to the same residues in PDZ2 of SAP97 383 in PDZ2 of SAP97. In 1-AR resulted in an energetically stable model where Val at P = 0 and Ser at P = −2 were bound to the expected residues of PDZ2. In addition, Lys323 and Lys326 were involved in stabilizing the binding pocket of PDZ2 as it transitions from the free to the liganded conformation 323 and Lys326 to alanine in PDZ2 of SAP97 abrogated the binding of PDZ2 to GluR1 and the ß1-AR, indicating that this pair of lysines played a prominent role in PDZ2-mediated interactions  indicating that there was a relationship between PDZ2-mediated inhibition of the binding between SAP97 and the ß1-AR and its effect on resensitization. After this determination, we proceeded to determine the effect of mutated PDZ2 (K323A/K326A) on resensitization of the ß1-AR. Our expectation was that overexpression of mutated PDZ2 would not affect the resensitization because the mutated PDZ2 did not bind to the ß1-AR (p<0.05 by one-way ANOVA) than those of WT PDZ2.Our hypothesis concerning PDZ2 was that if WT PDZ2 was expressed in >85% of the cells, it would compete effectively with endogenous SAP97 in binding to the ßhe ß1-AR . Resensi1-AR and SAP97 however, was not sufficient for mediating the functional effects of SAP97 on trafficking and on resensitization of the ß1-AR signaling pathway. This can be gleaned from the data of 1-AR and did not promote the recycling and externalization of internal ß1-AR. These data support the idea that SAP97 acted in part as a bridging molecule between the ß1-AR and I3-binding partners. One such binding partner is AKAP79/150, which upon binding to the ß1-AR-SAP97 complex targets PKA, PKC, and/or PP2B to the ß1-AR microdomain 1-AR The binding between the ß1-adrenergic receptosome, but the functional significance of these interactions has not been adequately discussed. We and other groups have identified several functional attributes to the ß1-AR/SAP97/AKAP79 scaffold. For example, this scaffold was involved in externalization and resensitization of the ß1-AR 1-AR PDZ domain in the carboxy tail in confining the ß1-AR to the plasma membrane of H9C2 cardiomyocyte-like cells 1-AR (via its PDZ) in a complex with SAP97 and AKAP79/150 was responsible for confining the ß1-AR in the cell membrane as measured by single-particle tracking microscopy 1-AR-SAP97 and AKAP79/150 in mouse cardiac myocytes  and to AKAP79 (via I3) recruited PKA to the ß1-AR. This model is similar to the model for targeting PKA to AMPAR through the association of the GluR1 subunit of AMPAR with SAP97 (via PDZ2) and with AKAP79 through SAP97 1-AR caused the activation of a pool of PKA in close proximity to the SAP97-GluR1 scaffold in perisynaptic membranes to faithfully phosphorylate GluR1 on Ser8451-AR and AMPAR could be brought into close proximity through their shared binding to PDZ2 of SAP97 homodimers that are formed by L27 domain oligomerization (2-AR via PDZ3 of PSD-95 was reported 2-AR and the complex of PSD-95-ß2-AR-AKAP79/150 was linked to GluR1 via stargazin to form a macromolecular complex between the ß2-AR and AMPAR. PSD-95 binds avidly to the ß1-AR PDZ domain, but unlike SAP97 the interaction between the ß1-AR and PSD-95 inhibited the internalization and recycling of the ß1-AR Thus far we have shown that SAP97 binding to the ßrization . Recentl