Does Internet-based guided-self-help for depression cause harm? An individual participant data meta-analysis on deterioration rates and its moderators in randomized controlled trials.

BACKGROUND: Almost nothing is known about the potential negative effects of Internet-based psychological treatments for depression. This study aims at investigating deterioration and its moderators within randomized trials on Internet-based guided self-help for adult depression, using an individual patient data meta-analyses (IPDMA) approach. METHOD: Studies were identified through systematic searches (PubMed, PsycINFO, EMBASE, Cochrane Library). Deterioration in participants was defined as a significant symptom increase according to the reliable change index (i.e. 7.68 points in the CES-D; 7.63 points in the BDI). Two-step IPDMA procedures, with a random-effects model were used to pool data. RESULTS: A total of 18 studies (21 comparisons, 2079 participants) contributed data to the analysis. The risk for a reliable deterioration from baseline to post-treatment was significantly lower in the intervention v. control conditions (3.36 v. 7.60; relative risk 0.47, 95% confidence interval 0.29-0.75). Education moderated effects on deterioration, with patients with low education displaying a higher risk for deterioration than patients with higher education. Deterioration rates for patients with low education did not differ statistically significantly between intervention and control groups. The benefit-risk ratio for patients with low education indicated that 9.38 patients achieve a treatment response for each patient experiencing a symptom deterioration. CONCLUSIONS: Internet-based guided self-help is associated with a mean reduced risk for a symptom deterioration compared to controls. Treatment and symptom progress of patients with low education should be closely monitored, as some patients might face an increased risk for symptom deterioration. Future studies should examine predictors of deterioration in patients with low education.

Red ear syndrome and auricular erythromelalgia: the same condition?

Several cases of relapsing attacks during which the ear becomes red and patients experience a burning sensation have been reported in the literature. This combination of symptoms has been described as 'red ear syndrome' (RES). We report on a 7-year-old boy who had episodes of reddening, swelling and a burning sensation in one ear with local hyperthermia persisting for 3 years. The differential diagnosis included RES and erythromelalgia, as isolated auricular variants of erythromelalgia have been described and the symptoms are difficult to distinguish from RES. In this report, we discuss the similarities and differences between RES and erythromelalgia.

[Drug therapy knowledge in ambulatory cardiovascular patients]. / Was wissen Herzkreislaufpatienten über ihre Medikamente?

To analyse drug therapy knowledge of prescribed drugs and self-administered medicinal products we interviewed 222 ambulatory cardiovascular patients. Spontaneous mentions of drugs (at least 3 preparations on average) were completed later on specific questioning. 40% of patients once interrupted their therapy due to adverse drug reactions. Most patients knew the labels of their medicaments but the effects and indications only in 50%. Nevertheless 70% were satisfied with the informations of the physician. Drugs available over the counter were consumed in many of the respondents: 50% consumed analgetics, and--mainly female patients--25% sedatives and 15% laxatives or diuretics. We conclude that specific questioning on self-administation of OTC preparations or formerly prescribed drugs results in an improved overview of medication and a better handling of polymedication and drug compliance.

The influence of floral display size on selfing rates in Mimulus ringens.

Pollinators often visit several flowers in sequence on plants with large floral displays. This foraging pattern is expected to influence the rate of self-fertilization in self-compatible taxa. To quantify the effects of daily floral display on pollinator movements and selfing, we experimentally manipulated flower number in four replicate (cloned) arrays of Mimulus ringens (Scrophulariaceae), each consisting of genets with unique combinations of homozygous marker genotypes. Four display classes (two, four, eight and 16 flowers) were present in each array. Pollinator visitation rate per flower and seed set per fruit were unaffected by display. However, flower number strongly influenced the frequency of within-plant pollinator movements, which increased from 13.8% of probes on two-flower displays to 77.6% of probes on 16-flower displays. The proportion of within-plant movements was significantly correlated with selfing (r = 0.993). The increase from 22.9% selfing on two-flower displays to 37.3% selfing on 16-flower displays reflects changes in the extent of geitonogamous self-pollination. We estimate that approximately half of all selfing on 16-flower displays resulted from geitonogamy. Selfing also varied dramatically among fruits within display classes. Nested ANOVA indicates that differences among flowers on two-flower ramets accounted for 45.4% of the variation in selfing, differences among genets accounted for 16.1% of the variation, and statistical and sampling error accounted for 38.5% of the variation. Differences among flowers within ramets may reflect the order of sequential floral probes on a display.

Subtype specific regulation of human vascular alpha(1)-adrenergic receptors by vessel bed and age.

BACKGROUND: alpha(1)-adrenergic receptors (alpha(1)ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (alpha(1a), alpha(1b), alpha(1 d)) mediating these effects is unknown and varies with species studied. In order to understand mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (as seen with aging), we tested two hypotheses: (1) human alpha(1)AR subtype expression differs with vascular bed, and (2) age influences human vascular alpha(1)AR subtype expression. METHODS AND RESULTS: Five hundred vessels from 384 patients were examined for alpha(1)AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel alpha(1)AR density is 16+/-2.3fmol/mg total protein. alpha(1a)AR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, alpha(1)AR mRNA subtype expression varies with vessel bed (alpha(1a) higher in splanchnic versus central arteries, P<0.05); competition analysis (selected vessels) and functional assays demonstrate alpha(1a) and alpha(1b)-mediated mammary artery contraction. Overall alpha(1)AR expression doubles with age (<55 versus > or = 65 years) in mammary artery (no change in saphenous vein), accompanied by increased alpha(1b)>alpha(1a) expression (P< = 0.001). CONCLUSIONS: Human vascular alpha(1)AR subtype distribution differs from animal models, varies with vessel bed, correlates with contraction in mammary artery, and is modulated by aging. These findings provide potential novel targets for therapeutic intervention in many clinical settings.

[Effect of smoking and antioxidant vitamin concentrations of pregnant patients on birth weight of newborn infants]. / Einfluss des Rauchverhaltens und der antioxidativen Vitaminkonzentrationen von Schwangeren auf das Geburtsgewicht von Neugeborenen.

An epidemiological study into the influence of cotinine validated maternal smoking and antioxidant vitamin concentrations on new born infants was carried out from 1992 to 1994 in the Department of Obstetrics and Gynaecology, University of Homburg/Saar. Of 222 participated pregnant women 26% were active smokers and 46% passive smokers. After adjustment a mean reduction in birth weight of 228 g was found in infants born to smokers, without dose correlation. Birth weight was not significantly reduced in infants born to passive smokers. There was no influence to the gestational age. A high correlation was established between maternal serum cotinine and umbilical cotinine (r = 0.91). In actively smoking mothers there was a positive correlation between vitamin E concentration and corrected infant birth weight (r = 0.33). Actively smoking mothers with children with a birthweight lower than the 25th percentile had significantly (p < 0.007) oftener lower vitamin E concentrations. Smoking increases the consumption of vitamin E, so that there is overproduction of peroxides and a reduction in prostacyclin. This lack of prostacyclin may lead to diminished perfusion of the placenta and may explain the birth weight reduction in smoker children.

Acute myocardial beta-adrenergic receptor dysfunction after cardiopulmonary bypass in patients with cardiac valve disease. Duke Heart Center Perioperative Desensitization Group.

BACKGROUND: Patients with cardiac valve disease (CVD) frequently have congestive heart failure (CHF) and chronic myocardial beta-adrenergic receptor (beta AR) desensitization. Cardiac surgery requiring cardiopulmonary bypass (CPB) is associated with increased plasma catecholamine concentrations, which might worsen myocardial beta AR function. We therefore tested the hypothesis that acute beta AR dysfunction occurs during CPB in patients with CVD. METHODS AND RESULTS: After informed consent, 50 patients were enrolled. Right atrial biopsy samples were obtained at initiation and conclusion of CPB to assess beta AR density and adenylyl cyclase (AC) activity. Plasma catecholamine concentrations increased 3-fold during CPB (P < 0.01). Although beta AR density remained constant, isoproterenol-stimulated AC activity decreased significantly (approximately 30%; P < 0.005). AC activity decreased 22% and 24% with direct G protein (NaF) or AC (manganese) activation, respectively. Patients with or without preoperative CHF exhibited similar degrees of acute myocardial beta AR dysfunction during CPB. CONCLUSIONS: Acute myocardial beta AR dysfunction occurs during CPB in patients with severe CVD requiring surgical correction, with or without preexisting CHF. The primary underlying mechanism involves functional uncoupling of the beta AR signal transduction pathway at the level of the AC moiety. This information should facilitate development of agents designed to prevent acute myocardial beta AR dysfunction during CPB, potentially leading to improved outcome in this high-risk population.

Acute depression of myocardial beta-adrenergic receptor signaling during cardiopulmonary bypass: impairment of the adenylyl cyclase moiety. Duke Heart Center Perioperative Desensitization Group.

BACKGROUND: Previously the authors showed that myocardial beta-adrenergic (betaAR) function is reduced after cardiopulmonary bypass (CPB) in a canine model Whether CPB results in similar effects on betaAR function in adult humans is not known. Therefore the current study tested two hypotheses: (1) That myocardial betaAR signaling is reduced in adult humans after CPB, and (2) that administration of long-term preoperative betaAR antagonists prevents this process. METHODS: After they gave informed consent, 52 patients undergoing aortocoronary surgery were enrolled. Atrial biopsies were obtained before CPB and immediately before discontinuation of CPB. Plasma catecholamine concentrations, myocardial betaAR density, and functional responsiveness (basal, isoproterenol, zinterol, sodium fluoride, and manganese-stimulated adenylyl cyclase activity) were assessed. RESULTS: Catecholamine levels increased significantly during CPB (P < 0.005). Myocardial betaAR adenylyl cyclase coupling decreased during CPB, as evidenced by a 21% decrease in isoproterenol-stimulated adenylyl cyclase activity (750 [430] pmol cyclic adenosine monophosphate per milligram total protein 15 min before CPB compared with 540 [390] at the end of CPB, P = 0.0062, medians [interquartile range]) despite constant betaAR density. Differential activation along the betaAR signal transduction cascade localized the defect to the adenylyl cyclase moiety. Administration of long-term preoperative betaAR antagonists did not prevent acute CPB-induced myocardial betaAR dysfunction. CONCLUSIONS: These data indicate that the myocardial adenylyl cyclase response to betaAR agonists decreases acutely in adults during aortocoronary surgery requiring CPB, regardless of whether long-term preoperative betaAR antagonists are administered. The mechanism underlying acute betaAR dysfunction appears to be direct impairment of the adenylyl cyclase moiety. Similar increases in manganese-stimulated activity before and at the end of CPB show preserved adenylyl cyclase catalytic activity, suggesting that other mechanisms (such as decreased protein levels or altered isoform expression or function) may be responsible for decreased adenylyl cyclase function.

Alpha1-adrenergic receptor subtypes in human detrusor.

PURPOSE: To identify and quantitate alpha1-adrenergic receptor (alpha1AR) subtype expression in human detrusor. MATERIALS AND METHODS: Initial studies to determine alpha1AR expression in human detrusor were performed using saturation binding with [125I]HEAT. Once the presence of alpha1ARs was documented, subtype (alpha1a, alpha1b, alpha1d) expression at the mRNA level (and comparison with rat) was determined with RNase protection assays (human detrusor) and RT-PCR (human detrusor, rat whole bladder). Competition binding analysis with the alpha1dAR-selective ligand BMY7378 was used to measure alpha1AR subtype expression at a protein level in human detrusor. RESULTS: Alpha1AR expression in human detrusor was low but reproducible (6.3 +/- 1.0 fmol./mg. total protein). RNase protection assays performed on total RNA extracted from human detrusor revealed the following alpha1AR subtype expression: alpha1d (66%) > alpha1a (34%), and no alpha1b. RT-PCR confirmed alpha1AR subtype mRNA distribution in human detrusor with alpha1d (approximately 60-70%) > alpha1a (approximately 30-40%), and a lack of alpha1b mRNA. Rat whole bladder expressed different alpha1AR subtype mRNA than human detrusor, with alpha1a approximately alpha1b approximately alpha1d. The presence of alpha1d > alpha1a expression in human detrusor was confirmed at a protein level by competition analysis utilizing BMY7378 which revealed a two-site fit, with Ki and high affinity binding (66%) consistent with the alpha1dAR subtype. CONCLUSIONS: Human detrusor contained two alpha1AR subtypes (alpha1d > alpha1a), a finding that is different from rat, another commonly used animal model. Since non-subtype selective alpha1AR antagonists ameliorate irritative bladder symptoms (in men and women with/without outlet obstruction), and Rec 15/2739 (alpha1a selective antagonist) does not improve symptom scores in BPH, our findings suggest bladder alpha1dARs may provide a potentially novel mechanism underlying these therapeutic benefits.

Quality of life in breast cancer survivors: implications for developing support services.

PURPOSE/OBJECTIVES: Quality of life (QOL) is becoming more important in regard to breast cancer as treatment advances extend the period of survivorship. The purpose of this article is to share the results of a cancer center's attempt to evaluate the QOL needs of breast cancer survivors in order to provide improved supportive-care services. DESIGN: Descriptive mailed survey. SETTING: A medical center in southern California. SAMPLE: A random sample of breast cancer survivors (N = 298). METHODS: Breast cancer survivors completed a mailed survey that included major outcome variables of QOL and pain. Subjects were stratified by three age groups: younger than 40 years, 40-60 years, and older than 60 years. MAIN RESEARCH VARIABLES: QOL subscales (physical, psychological, social, and spiritual well-being) and overall QOL score and pain as assessed by the Brief Pain Inventory. FINDINGS: Results indicated continued physical demands of breast cancer, including fatigue and pain, as well as psychological burdens related to fear of breast cancer recurrence and anxiety. The social well-being domain indicated some unique aspects of QOL when applied to breast cancer survivorship such as the fear of breast cancer in female relatives. The spiritual well-being domain illustrated the unique QOL aspects of life-threatening illness such as living with uncertainty and maintaining hope. Breast cancer survivors also reported positive aspects and life changes after successfully facing breast cancer. CONCLUSIONS: Breast cancer survivors experience many demands of illness across the physical, psychological, social, and spiritual domains. IMPLICATIONS FOR NURSING PRACTICE: The study's findings can be useful in directing cancer centers' efforts to provide comprehensive care for breast cancer survivors. Nurses play a critical role in leading these efforts for supportive-care services intended to improve the QOL of breast cancer survivors.

Quality of life in breast cancer. Part II: Psychological and spiritual well-being.

With an increasing number of women surviving breast cancer beyond treatment, the focus of care has shifted from the acute treatment-related side effects to long-term effects associated with changes in quality of life (QOL). Part I of this article described the impact of breast cancer on the domains of physical and social well-being of 21 long-term survivors of breast cancer through qualitative analysis. Part II explores the impact of breast cancer on the domains of psychological and spiritual well-being.

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Quality of life in breast cancer. Part I: Physical and social well-being.

Almost 2 million breast cancer survivors reside in the United States. An increase in consumer advocacy and media attention to this disease has helped bring breast cancer survivorship to the forefront of public attention. This has led to increased attention on quality of life (QOL) issues for these survivors of breast cancer. This two-part article presents the results of a qualitative, descriptive study evaluating the QOL of 21 breast cancer survivors. This study is based on our conceptual model of QOL including physical, psychological, social, and spiritual well-being. Part I of this article describes the impact of breast cancer on the physical and social domains of QOL based on in-depth interviews with breast cancer survivors.

Quality of life in breast cancer survivors as identified by focus groups.

Currently, over 1,700,000 women are living with breast cancer in the United States. These long-term survivors of breast cancer are challenged to redirect their energy from issues of cancer treatment and early side effects toward quality of life issues related to long-term survivorship, such as menopause, infertility, fear of recurrence, family distress, and uncertainty. In an attempt to obtain patient perspectives on quality of life and health care issues faced by breast cancer survivors, focus group methodology was utilized in the first year of a 2 year study. The sample was stratified to represent three age groups: < 40 years, 40-60 years, and > 60, and was intended to represent different developmental levels believed to have varying experiences with quality of life and potentially divergent needs following breast cancer diagnosis. Results of these focus groups revealed unique quality of life concerns of breast cancer survivors across four domains of physical, psychological, social, and spiritual well being. Each of these domains yields important implications for future research and clinical practice.

Quality of life in breast cancer.

PURPOSE: Currently, 1,721,700 women are living with breast cancer in the United States. As the number of survivors of breast cancer continues to rise, so must our knowledge about unique quality-of-life concerns. This article reports the results of a study on quality of life in women with breast cancer and validates the model of quality of life in this population. DESCRIPTION OF STUDY: To explore these concerns and to validate a breast cancer quality-of-life model, 21 survivors of breast cancer, across three age strata (younger than 40 years, 40 to 60 years, and older than 60 years), were interviewed and asked to complete quantitative surveys on pain and quality of life. RESULTS: Across all age groups, unique issues of survivorship include those related to physical, psychological, social, and spiritual well-being. In the domain of physical well-being, the areas of worst outcome were in menstrual changes and fertility, fatigue, and pain. In the domain of psychological well-being, predominant needs were in the areas of fear of the spread of cancer, distress from surgery, recurrence, fear of second cancer, impact on self-concept, and fear of future tests. The social well-being subscale identified the greatest disruption in the area of family distress. The spiritual well-being subscale showed greatest disruption in the area of uncertainty, although other aspects of this domain were usually rated in a positive direction (e.g., importance of religious activities). CLINICAL IMPLICATIONS: The data demonstrated the need for further research, assessment, and intervention across each of the quality-of-life domains. There is a significant need to address physical problems; however, the psychological domain demonstrated the greatest area of distress. The multidimensional needs of breast cancer survivors emphasize the need for multidisciplinary collaboration.

Growth hormone (GH) status regulates GH receptor and GH binding protein mRNA in a tissue- and transcript-specific manner but has no effect on insulin-like growth factor-I receptor mRNA in the rat.

The effect of growth hormone-deficiency (GHD) and treatment with recombinant bovine GH (bGH) or human IGF-I (hIGF-I) for 10 days on the expression of GH receptor (GHR), GH binding protein (GHBP) and of insulin-like growth factor-I receptor (IGF-IR) mRNA was examined using dw/dw and normal Lewis rats. Hepatic GHR and GHBP mRNA expression was significantly lower in dw/dw rats in comparison to Lewis rats (P < 0.01) while specific 125I-bGH binding to hepatic microsomal membranes was significantly higher (P < 0.01), suggesting a reduction in hepatic GHR turnover with GHD. Treatment with bGH reduced hepatic specific 125I-bGH binding in dw/dw rats, but had no effect in Lewis rats. Treatment with hIGF-I increased hepatic specific 125I-bGH binding in Lewis rats. Hepatic GHR and GHBP mRNA expression was not changed by bGH or hIGF-I treatment, suggesting that differences in hepatic specific 125I-bGH binding may be due to posttranscriptional mechanisms. GHBP mRNA expression was higher in kidney, heart, and muscle of dw/dw rats in comparison to Lewis rats (P < 0.01), while GHR mRNA abundance was not changed. Treatment of dw/dw rats with hIGF-I or bGH resulted in a coordinate reduction of GHR and GHBP mRNAs in kidney (P < 0.01). IGF-IR mRNA was not detected in liver and despite reduced plasma IGF-I levels and IGF-I mRNA expression IGF-IR mRNA abundance was not changed in nonhepatic tissues by GHD. Our data suggest that changes in plasma IGF-I levels and local IGF-I mRNA do not influence IGF-IR mRNA expression, while GHR and GHBP mRNA expression in different rat tissues are regulated independently. The increased nonhepatic GHBP mRNA expression with GHD suggests that nonhepatic GHBP may have an important physiological function distinct from that of GHBP in liver or in plasma.

The insulin-like growth factor-II/mannose-6-phosphate receptor is present in fetal bovine tissues throughout gestation.

The insulin-like growth factor-II (IGF-II) and the IGF-II/mannose-6-phosphate (M6P) receptor are thought to play an important role in fetal growth and development. We have studied the expression of the IGF-II/M6P receptor in fetal bovine tissues from 5 through 36 weeks' gestation. Tissues from bovine fetuses were extracted in buffer containing 2% Triton-X-100 and 2% sodium dodecyl sulfate (SDS). Aliquots of the protein extracts were analyzed by SDS polyacrylamide gel electrophoresis and the protein bands were transferred onto nitrocellulose. Immunoblotting was performed with anti-bovine IGF-II/M6P receptor antiserum. In a subset of experiments, ligand blotting was carried out with radiolabeled IGF-II and subsequent autoradiography. IGF-II/M6P receptors were expressed in all tissues examined, with the highest amount of receptor being present in fetal lung and liver. Low amounts of receptors were measured in fetal brain. The amount of receptor was developmentally regulated throughout fetal life. The developmental regulation of receptor expression varied among the different tissues. In conclusion, the IGF-II/M6P receptor is present in all fetal bovine tissues examined. The presence of the IGF-II/M6P receptor seems to be developmentally regulated during bovine fetal life. We hypothesize that this receptor exerts important biologic effects during fetal growth and tissue and organ development.

Characterization of ovine growth hormone (oGH) and ovine placental lactogen (oPL) binding to fetal and adult hepatic tissue in sheep: evidence that oGH and oPL interact with a common receptor.

The GH receptor (GHR) plays a key role in postnatal growth regulation. Although plasma concentrations of GH are high during fetal life, its role during fetal development is not well understood. Recent data suggest that GHR are present in fetal hepatic tissue as early as 51 days gestation. However, the levels of GHR expression are markedly lower in fetal hepatic tissue compared to postnatal values, and there are conflicting data suggesting that ovine placental lactogen (oPL) and oGH share a common receptor. Given the uncertainty about whether oPL acts via the oGHR or a distinct receptor, we performed ligand binding and affinity cross-linking studies on hepatic microsomal membranes from adult castrated male, pregnant female, and fetal sheep. Ligand binding assays at a constant concentration of membranes showed that [125I]oPL yielded consistently higher (P < 0.001) specific binding (59.5 +/- 6.4%, 30.5 +/- 5.7%, and 7.6 +/- 2.4% for castrated male, pregnant female, and fetal sheep, respectively) compared to [125I]oGH (17.8 +/- 4.7%, 5.0 +/- 1.6%, and 1.2 +/- 0.4% for castrated male, pregnant female, and fetal sheep, respectively). Cross-reactivity studies showed that unlabeled oPL was consistently more potent than unlabeled oGH in displacing either of the labeled ligands. The dissociation constant (Kd) for oPL binding ranged from 0.16-0.40 nM and was not changed by solubilization with Triton X-100. Equilibrium binding analysis for oGH showed lower affinity for hepatic microsomal membranes (Kd, 1.7-3.2 nM) in each of the three groups of animals. Affinity cross-linking of microsomal membranes from castrated male and pregnant female sheep liver showed four major cross-linked complexes with both [125I]oPL and [125I]oGH, with mol wt of 150, 97, 75, and 60 kilodaltons. All four bands were identified with both ligands. Unlabeled oPL showed markedly higher potency than unlabeled oGH in reducing the signal of the [125I]oPL cross-linked complexes, whereas unlabeled oGH and oPL showed comparable potencies in reducing the signal of the [125I]oGH complexes. Immunoprecipitation of detergent-solubilized hepatic microsomal membranes from pregnant and fetal sheep using a panel of monoclonal antibodies raised against the extracellular region of the rabbit GHR showed potent immunological recognition of the [125I]oPL-receptor complexes. We suggest that oGH and oPL bind to a common or a related receptor protein(s). It is possible that differences in receptor dimerization or association with other membrane proteins are the basis of the differences in affinity and biological actions of the two hormones.

Perturbation of C6 glial cells by acetate leads to modulation of [125I]IGF-II binding to the IGF-II/M6P receptor: implications for ligand internalization studies.

Acid washes are used as an experimental tool to differentiate between cell-surface bound and internalized radioligands. We have observed that washes with acid buffers containing 100 mM acetate can modulate [125I]IGF-II binding to rat C6 glial cells in an unexpected manner: when cells in monolayer culture were prewashed with phosphate buffered saline (pH 7.3) (PBS), [125I]IGF-II binding was characteristic of the IGF-II/mannose-6-phosphate (M6P) receptor. Importantly, IgG 3637, which is purified from an antiserum directed against the rat IGF-II/M6P receptor, blocked binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity crosslinking studies using DSS as the crosslinking agent and Western blotting experiments using antiserum 3637 confirmed the presence of the IGF-II/M6P receptor in C6 glial cells. Prewashes of C6 cell monolayers with acid buffers (pH 4-4.5) which contained 100 mM sodium acetate and which have been used in internalization studies reduced [125I]IGF-II binding by 40-60%. Affinity crosslinking studies using C6 cells showed that the formation of the 250 kDa radioligand-receptor complex was not prohibited by IgG 3637 after acid washes with buffers containing high acetate concentrations, while acid washes with buffers containing no acetate did not cause a loss in the blocking ability of IgG 3637. However, acid washes with 100 mM acetate did not alter the recognition of IGF-II/M6P receptors by IgG 3637 in Western blotting experiments. In addition, in a subset of experiments acid prewashes with acetate also decreased binding of [125I]IGF-I to the IGF-I receptor by 20%. We conclude that acid washes with acetate buffers lead to decreased [125I]IGF-I and [125I]IGF-II binding. In addition, the capability of anti-receptor IgG to block radioligand binding to the IGF-II/M6P receptor also declines. We hypothesize that alteration of ligand binding might be partially caused by perturbation of the cell membrane and hence a conformational change in IGF receptors. These data imply that the use of acetate buffers in acid wash experiments in ligand internalization studies--particularly in studies involving the IGF-II/M6P receptor--should be avoided.