The Attractions of Proteins for Small Molecules and Ions

Scatchard, George

doi:10.1111/j.1749-6632.1949.tb27297.x

Cited by 19,706 publications

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References 13 publications

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“…Maximal saturation of the antagonist occurred at a concentration of 0.2nM. Scatchard analysis [30] indicates that the maximum number of binding sites is 23.1 fmol/dish (21 fmol/mg protein) and the K d for [ 3 H]CPX is 0.13 nM (Fig. 3, inset), which is in adequate agreement with the k d value calculated from the kinetic experiments.…”

supporting

confidence: 81%

Characterization of adenosine receptors in intact cultured heart cells

El-Ani

Shainberg

1994

Biochemical Pharmacology

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Adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. We used the hydrophilic A 1 adenosine receptor radioligand [ 3 H]-8-cyclopentyl-1,3-dipropylxanthine ([ 3 H]CPX), to monitor the level of the receptors on intact cardiocytes. The binding showed high affinity (K d = 0.13 nM) and the number of [ 3 H]CPX binding sites (B max ) was 23.1 fmol/dish (21 fmol/mg protein). The K i of the agonists R-N 6 -(2-phenylisopropyl)-adenosine (R-PIA) and S-N 6 -(2-phenylisopropyl)-adenosine (S-PIA), and of the antagonists CPX and theophylline were 3.57, 49.0, 1.63 and 4880 nM, respectively. The number of adenosine receptors was very low during the first days in cultures (5 fmol/dish) and increased gradually until it reached a plateau on days 8-10. Treatment with norepinephrine or isoproterenol which accelerated the rate of contractions, induced up regulation of the receptors. B max increased 2-3-fold by application of norepinephrine for 4 days, while receptor affinity to the radioligand was unaffected. Lactate dehydrogenase (LDH) and creatine kinase (CK) activity increased only by 22 and 38%, respectively. Similarly, 3 days treatment with triiodothyronine (T 3 , 10 −8 M), which also accelerated heart rate, increased the number of adenosine receptors by 56% without a significant change in the affinity of the receptors to [ 3 H]CPX. Carbamylcholine (5 × 10 −6 M), which reduced the rate of heart contractions, caused 26% down regulation while the affinity of the receptors remained unchanged. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, the level of adenosine receptors may respond to drug-induced chronic changes in cardiac contractile activity so as to restore conditions to normal (basal) contractions. KeywordsCPX; heart-rate; norepinephrine; thyroid hormones Adenosine modulates a variety of physiological functions in the heart. These actions are mediated by cell surface adenosine receptors, which can be classified into A 1 and A 2 † Corresponding author. Tel. (972)3-5318265; FAX (972)3-5351824. In conditions of stress like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP [4]. In these conditions adenosine has therapeutic and protective effects on the heart [5,6]. Adenosine causes negative chronotropic, dromotropic and ionotropic effects on the cardiac tissue via A 1 receptors [7,8]. Adenosine also acts as a coronary vasodilator via A 2 receptors [9]. These effects of adenosine occur also without prior catecholamine treatment [10]. Stimulation that enhances the cAMP content (catecholamines, forskolin, amrinone), makes the heart more sensitive to adenosine [11][12][13][14]. In addition, adenosine causes hyperpolarization by activation of K + channels [15] via G proteins [16, 17]. These K + channels are the same channels that are stimulated by acetylcholine in the cardiac tissue [18][19][20]. HHS Public AccessChron...

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confidence: 81%

Characterization of adenosine receptors in intact cultured heart cells

El-Ani

Shainberg

1994

Biochemical Pharmacology

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“…Dextran-coated charcoal suspension was used to separate the bound and free fractions. The receptor concentration was calculated by Scatchard analysis (Scatchard, 1949).…”

Section: Methodsmentioning

confidence: 99%

Oestrogen receptors and the response to endocrine therapy in advanced breast cancer

Roberts¹,

Rubens²,

King³

et al. 1978

Br J Cancer

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Summary.-The relationship between oestrogen-receptor protein (ER) content of the tumour and the response to endocrine therapy was determined in 119 patients, in a collaborative prospective study. Twenty-eight of the 80 patients with measurable ER responded to treatment according to UICC criteria, compared with only 3/39 without ER. It was found that site of biopsy did not influence the result, but tumour content of the tissue sample was significantly related to the presence of receptors. The organizational problems of such a study are discussed.RESULTS from a number of centres in the United States and Europe indicate that the response of advanced breast cancer to endocrine therapy can be correlated with the oestrogen-receptor content of the tumour (McGuire et al., 1975). In 1974 the British Breast Group initiated a study to investigate these reports in a prospective series.Two centres took part in our study, both referring all patients suitable for endocrine therapy over a 30-month period (1.10.74-31.3.77). The clinicians were not informed of the results of oestrogen-receptor assays, and patients were treated according to the routine protocols for each centre. Our study included external clinical assessment of response to therapy, pathological review of all biopsy specimens, and a comparison of laboratory methodology. METHODSCriteria for inclusion.-Consecutive unselected patients from Guy's Hospital and the Royal Infirmary of Edinburgh breast clinics were included in this study, provided they had evidence of progressive, histologically confirmed, locally advanced or disseminated breast cancer, and had a lesion accessible for biopsy for the estimation of oestrogenreceptor content. Most patients had not received previous systemic therapy for advanced disease, but in the event of previous additive hormone therapy, at least 14 days were allowed to elapse before biopsy was performed. Biopsy, from a single site, was carried out immediately before new endocrine therapy was started.Pathological assessment.-Sections from every biopsy specimen were examined by one pathologist (R.R.M.) without knowledge of the oestrogen-receptor assay result, to determine whether adequate tumour tissue was present. An 8-point scoring system was devised as follows:(a) the overall proportion of neoplastic tissue to normal tissue in the section was allocated a score of 1 (<50 /) to 4 (100%) (b) the proportion of malignant epithelial cells to stroma within the neoplasm was scored 1 (<50%) to 4 (100%).

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“…A portion (160 >mg) of fat-free tumour was homogenized in tris buffer (0-25 M sucrose, 10 mM tris and 1 mM ethylene diamine tetra-acetate) at the rate of 100 mg/ml for 3 x 15 s, with intervals for cooling between periods of homogenization. (0, 10, 30, 50, 70, 90, 20,000 pg) were calculated from a Scatchard (1949) analysis of the data. The concentration of oestrogen receptors in each tumour sample was also expressed on a protein basis (PO, protein = fmol/mg extracted protein).…”

Section: Methodsmentioning

confidence: 99%

Reproducibility of measurements of oestrogen-receptor concentration in breast cancer

Hawkins¹,

Hill²,

Freedman³

et al. 1977

Br J Cancer

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Summary.-The reproducibility of measurements of oestrogen-receptor activity has been examined in multiple specimens from a rabbit uterus, a rat mammary tumour and human breast tumours. The relationship between receptor concentration and tumour histology has also been investigated in 11 large primary tumours.In the animal tissues, receptor measurements were relatively reproducible (co- In the 11 primary breast cancers selected for study, the level of receptor activity was related to menopausal status and the tumour content of the specimen.We conclude that the receptor activity detected varies within a tumour and depends upon the tumour content of the biopsy specimen. Predictions based on precise quantitation of receptor concentrations may therefore necessitate replicate tumour sampling and correction for the fraction of non-tumour tissue in each sample.CURRENTLY, the best index of the hormonal sensitivity of a breast cancer is the concentration of oestrogen-receptor protein in the cytoplasm of the tumour. (Folca, Glascock and Irvine 1961;Jensen et al., 1971;McGuire et al., 1975). We have, therefore, investigated the reproducibility of receptor measurements and the role of morphological factors in determiining variations in receptor concentration. MATERIALS AND METHODSTissues.-To examine the precision of the receptor assay, multiple (4-8) portions were cut at 0-40C, from each of 4 tissues selected for their apparent homogeneity. These tissues 'were the uterus from a non-pregnant rabbit, a rat mammary carcinoma (generated by the intragastric administration of 30 mg dimethylbenz(a)anthracene at 50 days of age) a large, cellular, intracanalicular fibroadenoma removed surgically from a 59-year-old woman, and a lymplh node which had been largely replaced by anaplastic breast carcinoma from a 75-year-old woman. Each portion of tissue was assayed for oestrogen-receptor activity.To examine the reproducibility of receptor measurements in human breast cancer an(d relate this to morphology, 11 mastectomy specimens containing large, primary breast cancers were collected on ice. Each tumour wTas excised from the breast, measured and sectioned into 2-3 cubes. Each cube was divided into 2, one portion being used for histological examination, the other for oestrogen-receptor assay.Oe8trogen-receptor activity was determined by the method of Hawkins, Hill and Freedman (1975). A portion (160 >mg) of fat-free tumour was homogenized in tris buffer (0-25 M sucrose, 10 mM tris and 1 mM ethylene diamine tetra-acetate) at the rate of 100 mg/ml for 3 x 15 s, with intervals for cooling between periods of homogenization.

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The Attractions of Proteins for Small Molecules and Ions

Cited by 19,706 publications

References 13 publications

Characterization of adenosine receptors in intact cultured heart cells

Characterization of adenosine receptors in intact cultured heart cells

Oestrogen receptors and the response to endocrine therapy in advanced breast cancer

Reproducibility of measurements of oestrogen-receptor concentration in breast cancer

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