Gender differences with short-term vs 12 months dual antiplatelet therapy in patients with acute coronary syndrome treated with the COMBO dual therapy stent: 2-years follow-up results of the REDUCE trial.

BACKGROUND: Gender differences in the thrombotic and bleeding risk have been suggested to condition the benefits of antithrombotic therapies in Acute Coronary Syndrome (ACS) patients, and mainly among those undergoing percutaneous coronary interventions with drug eluting stents (DES). The impact of gender on the optimal duration of dual antiplatelet therapy (DAPT) in ACS patients is still unexplored and was, therefore, the aim of the present sub-study. METHODS: REDUCE was a prospective, multicenter, randomized investigator-initiated study designed to enroll 1500 ACS patients after treatment with the COMBO Dual Stent Therapy, based on a noninferiority design. Patients were randomized in a 1:1 fashion to either 3 or 12 months of DAPT. Primary study endpoint was a composite of all-cause mortality, myocardial infarction, definite/probable stent thrombosis (ST), stroke, target-vessel revascularization (TVR) and bleedings (BARC II, III, V) at 12 months. Secondary endpoints were cardiovascular mortality and the individual components of the primary endpoint within 24 months. RESULTS: From June 2014 to May 2016 300 women and 1196 men were included in the study. Among them, 43.7% of females and 51.9% of males were assigned to the 3 months DAPT treatment. Baseline characteristics were well matched between the two arms, with the exception of a lower rate of TIMI flow < 3 (p = 0.04), lower systolic blood pressure (p = 0.05) and use of spironolactone (p = 0.006) among women and a more advanced age (p = 0.05) among men receiving a short-term DAPT. At a mean follow-up of 525 (± 198) days, no difference in the primary endpoint was observed according to DAPT duration in both females [6.9% vs 5.9%, HR (95% CI) = 1.19 (0.48-2.9), p = 0.71] and males [8.2% vs 9%, HR (95% CI) = 0.92 (0.63-1.35), p = 0.67; p INT = 0.20]. Results were confirmed after correction for baseline differences [females: adjusted HR (95% CI) = 1.12 (0.45-2.78), p = 0.81; males: adjusted HR (95% CI) = 0.90 (0.61-1.32), p = 0.60]. Comparable rates of survival, thrombotic (MI, stent thrombosis, TVR, stroke) and bleeding events were observed with the two DAPT strategies, with no impact of gender. CONCLUSIONS: The present study shows that among ACS patients randomized in the REDUCE trial, a 3 months DAPT strategy offers comparable results as compared to a standard 12 months DAPT at 2-years follow-up in both male and female gender.

The development of an ICF-based questionnaire for patients with chronic conditions in primary care.

OBJECTIVES: We developed a self-reported questionnaire for patients in primary care with chronic conditions aged 50 years or older. The questionnaire supports a more person-centered approach by adopting a biopsychosocial focus on functional status instead of a focus on disease. STUDY DESIGN AND SETTING: Based on the International Classification of Functioning, Disability and Health (ICF), an ICF Primary Care set for patients with chronic conditions was constructed in three phases. In the first phase, we identified relevant ICF categories for the ICF Primary Care set by using existing ICF sets for chronic health conditions. The ICF Primary Care set was completed by a multidisciplinary expert panel and consisted of 52 ICF categories covering ICF's body functions, activities, participation, environmental factors, and personal factors. In the last phase, we constructed a draft version of the questionnaire by converting the ICF categories from the ICF Primary Care set into questions and corresponding scales. To improve the draft version of the questionnaire, we conducted cognitive interviews with patients with chronic conditions in an iterative process, focusing on the problems patients experienced in answering the items of the questionnaire. Interview analysis was used for assessing the content and construct validity of the questionnaire. RESULTS: Thirty cognitive interviews with patients were conducted in five different interview rounds. In these interviews, we identified 124 problems in the responding process of answering the questionnaire, mostly concerning difficulties with the comprehension of the constructs of the questions. The number of problems reduced from an average of 11 problems per interview in the first round to an average of two problems in the last round. CONCLUSION: Conclusion: The final version of the questionnaire demonstrated high content and construct validity (i.e., patients are well capable of describing their functional status in terms of ICF) and is applicable in primary care in the Netherlands.

[Approaching psychological problems like a general practitioner]. / Psychische problemen benaderen als een huisarts.

An important cause of the high prescription levels of psychotropic medication for psychological symptoms is that these symptoms are assessed according to the same model as applied for physical symptoms, the disease model. This has led to a one-sided medical approach to psychological symptoms. A person-centred approach offers an alternative; the positive aspects of the disease-centred approach are retained and attention for the patient and his/her context become the central focal point for the general practitioner. Important elements of the person-centred approach are empathy, a good doctor-patient relationship, a shared approach to problem definition and understanding of the patient's problem, development of a therapeutic alliance, and a focus on the patient's hopes and expectations. If additional primary care-based treatment by mental health practice nurses is indicated, this model could be suitable since it is based on patients' strengths and focuses on personal growth rather than reduction of symptoms.

[A woman with excessive dynamic airway collapse]. / Een vrouw met 'excessive dynamic airway collapse'.

BACKGROUND: 'Excessive dynamic airway collapse' (EDAC) is a form of tracheamalacia, that does not affect the rings of cartilage on the anterior side of the trachea, but in which the posterior wall of the trachea collapses during inspiration and expiration. CASE DESCRIPTION: An 80 year-old woman presented to the accident and emergency department with dyspnoea and severe inspiratory and expiratory stridor, with sudden onset a few hours previously. Following a series of investigations, she was shown to have a narrowing of the trachea, radiating from the posterior wall. The diagnosis was EDAC. Because her saturation levels were acceptable and she was otherwise well she was admitted to the intensive care unit for observation only. The symptoms disappeared within 4 days, without intervention. CONCLUSION: EDAC is usually asymptomatic, but when symptoms arise the clinical picture is often confused with asthma, COPD or laryngeal oedema. The diagnostic gold standard is bronchoscopy. Therapy is chosen on the basis of severity of the symptoms.

[A young man with a painful knee]. / Een jongeman met een pijnlijke knie.

A 23-year-old man injured his left knee. A CT scan showed a dislocation of the proximal tibiofibular joint. Closed reduction of the dislocation was done successfully under procedural sedation and analgesia. Afterwards he was treated with a pressure bandage and immobilisation of the knee for 2 weeks.

The extent of ST elevation and ST deviation as predictors of mortality in ST-segment elevation myocardial patients planned to undergo primary percutaneous coronary intervention.

BACKGROUND: Only few data are available on the predictive value of ST deviation (both ST elevation and depression). Therefore, we have examined the predictive value of ST elevation and ST deviation in STEMI patients on 30-day and long term mortality. METHODS: All STEMI patients with an interpretable diagnostic electrocardiogram, who were referred to the Isala hospital and were planned to undergo a primary coronary intervention (pPCI) in the period 2001 until 2009, were prospectively registered in a cohort study. These patients were divided in tertiles based on the cumulative (cum) ST deviation (D1, D2 and D3) and cum ST elevation (E1, E2 and E3), as assessed by an independent core-lab. RESULTS: In total, 4513 patients were registered. 30-day mortality increased with cum ST deviation (0-9 mm: 1.9%, > 9-16 mm: 2.4%, > 16 mm: 3.9%, P = 0.001), but not significant with cum ST elevation. Long term mortality increased with cum ST-deviation (0-9 mm: 18.6%, > 9-16 mm: 22.1%, > 16 mm: 25.7%, P < 0.001) and with cum ST-elevation (0-6mm: 19.7%, > 6-11 mm: 22.7%, > 11 mm: 24.2%, P = 0.070). After multivariable adjustment using Cox proportional Hazard models, cum ST deviation (D1: reference, D2: HR: 1.09 95% CI (0.67-1.77), D3: HR: 1.76 95% CI (1.14-2.73)) was independently associated with 30-day mortality. Both cum ST deviation (D1: reference, D2: HR: 1.14 95% CI (0.98-1.34), D3: HR: 1.32 95% CI (1.13-1.53)) and ST elevation (E1: reference, E2: HR: 1.17 95% CI (1.00-1.38), E3: HR: 1.21 95% CI (1.04-1.42)) were independently associated with long term mortality. CONCLUSIONS: Besides ST elevation, taking the extent of ST depression into account improves the predictive value of the diagnostic 12 lead electrocardiogram especially for 30-day mortality in STEMI patients who are planned to undergo pPCI.

Decision-making in female fertility preservation is balancing the expected burden of fertility preservation treatment and the wish to conceive.

STUDY QUESTION: What are the decisive factors in fertility preservation (FP) decision-making in young women scheduled for gonadotoxic therapy? SUMMARY ANSWER: FP decision-making in young women scheduled for gonadotoxic therapy is mainly based on weighing two issues: the intensity of the wish to conceive a child in the future and the expected burden of undergoing FP treatment. WHAT IS KNOWN ALREADY: Future fertility is of importance for young cancer patients whose reproductive function is being threatened by oncological therapy. To prevent or reduce severe psychological effects of infertility as well as feelings of regret about their FP decision after cancer treatment, the quality of fertility preservation counselling (FPC) should be improved. To improve care, those issues forming a decisive factor in FP decision-making for patients should be clarified, as these issues deserve extensive discussion during FPC. Until now, decisive factors have not been isolated from the complex interplay of all aspects of FP that women contemplate during FP decision-making. STUDY DESIGN, SIZE, DURATION: By using a mixed methods methodology, a questionnaire developed after qualitative research involving a selected group of five women who previously received FPC was retrospectively sent to eligible patients (n = 143) who had received FPC (1999 - July 2013) and to whom at least one FP option was offered. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients had received FPC at a university hospital in the Netherlands, in a setting where financial factors do not play a role in FP. They were aged ≥16 years and were scheduled for gonadotoxic treatment. The relationship between patients' baseline characteristics, their attributed importance to 28 relevant importance items and their FP choices was investigated. MAIN RESULTS AND THE ROLE OF CHANCE: After five interviews, 28 importance items for FP decision-making were identified and included in our questionnaire. Of these 28 importance items, 24 items could be clustered into seven importance themes. A total of 87 patients (61%) responded to our questionnaire. After performing a multivariable logistic regression analysis, proceeding with FP was related to higher attributed importance during FP decision-making to the theme 'Wish to conceive (in the future)' (odds ratio (OR) 10.8, 95% confidence interval (CI) 3.5-34.4) and the item 'Having a stable partner relationship' (OR 2.0, 95% CI 1.0-4.1), while higher attributed importance to the theme 'Expected burden of FP' during FP decision-making (OR 0.08, 95% CI 0.02-0.3) more often resulted in refraining from treatment. LIMITATIONS, REASONS FOR CAUTION: Besides possible recall and selection bias, the fact that this study was performed in Dutch patients aged ≥16 years counselled in a single centre, where finance was not an additional consideration, possibly limits the generalizability of our results to a broader European population of cancer patients. Furthermore, we are not able to draw conclusions about the causality of the associations observed in our study. WIDER IMPLICATIONS OF THE FINDINGS: The wish to conceive and the expected burden of FP treatment should be discussed carefully with patients during FP decision-making, either by the referring healthcare provider or by reproductive medicine specialist. Prospective research is needed to explore the causality of the associations found in this study. Furthermore, in order to deliver high quality patient-centred care, the development of tools to explore patients' wish to conceive (for example in different age categories) and tools to provide clear information about the burden of FP treatments (using the preferred information channels suggested by patients) is needed. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Radboud Institute for Health Sciences (research school affiliated to the Radboud university medical center). The authors have declared no conflicts of interest with respect to this work.

The influence of residential distance on time to treatment in ST-elevation myocardial infarction patients.

AIMS: To evaluate the relation between residential distance and total ischaemic time in patients with acute ST-elevation myocardial infarction (STEMI). METHODS: STEMI patients were transported to the Isala Hospital Zwolle with the intention to perform primary percutaneous coronary intervention PCI (pPCI) from 2004 until 2010 (n = 4149). Of these, 1424 patients (34 %) were referred via a non-PCI 'spoke' centre ('spoke' patients) and 2725 patients (66 %) were referred via field triage in the ambulance (ambulance patients). RESULTS: A longer residential distance increased median total ischaemic time in 'spoke' patients (0-30 km: 228 min, >30-60 km: 235 min, >60-90 km: 264 min, p < 0.001), however not in ambulance patients (0-30 km: 179 min, >30-60 km: 175 min, >60-90 km: 186 min, p = 0.225). After multivariable linear regression analysis, in 'spoke' patients residential distance of >30-60 km compared with 0-30 km was not independently associated with ischaemic time; however, a residential distance of >60-90 km (exp (B) = 1.11, 95 % CI 1.01-1.12) compared with 0-30 km was independently related with ischaemic time. In ambulance patients, residential distance of >30-60 and >60-90 km compared with 0-30 km was not independently associated with ischaemic time. CONCLUSION: A longer distance from the patient's residence to a PCI centre was associated with a small but significant increase in time to treatment in 'spoke' patients, however not in ambulance patients. Therefore, referral via field triage in the ambulance did not lead to a significant increase in time to treatment, especially at long distances (up to 90 km).

Early ambulance initiation versus in-hospital initiation of high dose clopidogrel in ST-segment elevation myocardial infarction.

Pre-hospital infarct diagnosis gives the opportunity to start anti-platelet and anti-thrombotic agents before arrival at the PCI centre. However, more evidence is necessary to demonstrate whether high dose (HD) clopidogrel (600 mg) administered in the ambulance is associated with improved initial patency of the infarct related vessel (IRV) and/or clinical outcome compared to in-hospital initiation of HD clopidogrel. From 2001 until 2009 all consecutive ST-Segment Elevation Myocardial Infarction (STEMI) patients who underwent pre-hospital diagnosis and therapy in the ambulance were prospectively included in our single-centre cohort study. We compared initial patency of the IRV and clinical outcome in patients treated from 2001 until June 2006 (in-hospital HD clopidogrel) with patients treated from July 2006 until 2009 (ambulance HD clopidogrel). A total of 2,475 patients with STEMI were registered; of these 1,110 (44.8%) received in-hospital HD clopidogrel and 1,365 (55.2%) received ambulance HD clopidogrel. Ambulance HD clopidogrel was not independently associated with initial patency (TIMI-2/3-flow pre-PCI (odds ratio: 1.18, 95% confidence interval [CI] 0.96-1.44); however, it was associated with fewer recurrent myocardial infarctions at 30 days (hazard ratio [HR]: 0.45, 95% CI 0.22-0.93) and at one year (HR: 0.45, 95% CI 0.25-0.80). No difference in TIMI 2/3 flow post-PCI, major bleeding, mortality, MACE - and the combination of mortality and recurrent myocardial infarction at 30-days and at one year was present between the two groups. In conclusion, early in-ambulance as compared to in-hospital initiation of HD clopidogrel in STEMI patients did not improve initial patency of the IRV or clinical outcome, except for a reduction of recurrent myocardial infarction. Therefore, early administration of HD clopidogrel seems to have net clinical benefit for these patients.

Application of spectral phase shaping to high resolution CARS spectroscopy.

By spectral phase shaping of both the pump and probe pulses in coherent anti-Stokes Raman scattering (CARS) spectroscopy we demonstrate the extraction of the frequencies, bandwidths and relative cross sections of vibrational lines. We employ a tunable broadband Ti:Sapphire laser synchronized to a ps-Nd:YVO mode locked laser. A high resolution spectral phase shaper allows for spectroscopy with a precision better than 1 cm(-1) in the high frequency region around 3000 cm(-1). We also demonstrate how new spectral phase shaping strategies can amplify the resonant features of isolated vibrations to such an extent that spectroscopy and microscopy can be done at high resolution, on the integrated spectral response without the need for a spectrograph.

Safety of citrate based hemofiltration in critically ill patients at high risk for bleeding: a comparison with nadroparin.

PURPOSE: To study the incidence and severity of bleeding in high risk critically ill patients during high volume, citrate based continuous veno-venous hemofiltration (CVVH). DESIGN: A prospective 1-year observational cohort study comparing citrate based CVVH with nadroparin based CVVH. PROCEDURES: Critically ill patients with multiple organ dysfunction and in need of CVVH were observed for bleeding complications during their CVVH sessions. Pre-defined criteria determined that patients were treated with citrate based CVVH in case of active bleeding or increased risk for bleeding. Otherwise nadroparin was used as anticoagulant. Statistical and outcome methods: The incidence of bleeding complications, the number of transfused blood cell concentrates and the filter-run-time were recorded. Analyses were made by non-parametric tests. MAIN FINDINGS: Fifty-five patients received 272 CVVH sessions. In the citrate group 14.8% experienced a bleeding complication compared to 25% in the nadroparin group (p=0.04). The number of transfused red blood cell concentrates was not different between groups. The nadroparin group had a longer filter run time (median 31.5 hours versus 22.5 hours, p=0.0001). CONCLUSIONS: In high risk critically ill patients citrate based anticoagulation for CVVH is safe in terms of bleeding complications and transfusion requirements.

Outcomes of adults with acute myelogenous leukemia in remission given 550 cGy of single-exposure total body irradiation, cyclophosphamide, and unrelated donor bone marrow transplants.

On the basis of observations from dog models and human studies, we hypothesized that a low-dose (550 cGy), single-exposure total body irradiation (TBI)-based regimen would result in improved survival when given to adult patients with acute myelogenous leukemia (AML) who were undergoing unrelated donor bone marrow transplantation in complete remission (CR). The regimen consisted of single exposure (550 cGy) of TBI given at a high dose rate (30 cGy/min) and cyclophosphamide. Graft-versus-host disease prophylaxis consisted of cyclosporine, methotrexate, and corticosteroids. Thirty-two consecutive adult patients (median age, 47 years) with AML in CR (15 in CR 1 and 17 in CR > or =2) were treated. Sixteen patients (50%) were alive and in remission at last follow-up (median, 2.2 years; range, 0.6-4.0 years). Kaplan-Meier estimates of overall and leukemia-free survival at 3 years were 55% +/- 14% (mean +/- SE) and 57% +/- 14% in CR 1 patients and were both 39% +/- 12% in CR > or =2 patients. Transplant-related mortality was 13% for patients in CR 1 and 41% for those in CR > or =2. Only 1 patient (3%) experienced fatal regimen-related organ toxicity, and only 1 had grade III or IV acute graft-versus-host disease. Graft failure was not observed. Relapse occurred in 22% of patients. This low-dose (550 cGy), single-exposure TBI-based regimen resulted in good survival and a low risk of fatal regimen-related organ toxicity in adult patients with AML who underwent unrelated donor bone marrow transplantation in CR.

Gap junctions in the rabbit sinoatrial node.

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.

Tumour necrosis factor alpha alters the expression of connexin43, connexin40, and connexin37 in human umbilical vein endothelial cells.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in orchestrating inflammatory responses with the vascular endothelium as main target cell type, and was found to promote migration of endothelial cells, as occurs in wound healing processes. Substantial evidence exists that endothelial cell migration in wound healing is related to changes in cell coupling by means of gap junctions. Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between cells. The authors have investigated whether TNF-alpha alters the expression of gap junction proteins (connexins, Cx) between human umbilical vein endothelial cells (HUVEC), thereby changing the extent of intercellular communication, as measured by dye coupling. Under control conditions, Cx43, Cx40, and Cx37 protein and mRNA were present in HUVEC. After exposure to 0.5 nM TNF-alpha for 48 h, however, the authors were no longer able to detect Cx37 and Cx40 protein, whereas Cx43 levels seemed unaltered but showed more perinuclear staining. After 24 and 48 h exposure to TNF-alpha, levels of Cx37 and Cx40 mRNA, were reduced, while the level of Cx43 mRNA remained unaltered, suggesting transcriptional regulation. If TNF-alpha was removed from the medium, Cx37 and Cx40 expression was restored within 24 h. The modulation of connexin expression by TNF-alpha resulted in a decrease in dye coupling of 40%.

Recovery from cytomegalovirus infection is associated with activation of peripheral blood lymphocytes.

The number of CD8bright and CD56+ lymphocytes in the peripheral blood and their activation status were monitored by flow cytometry in 23 renal transplant recipients with cytomegalovirus (CMV) infection and were correlated with the virus load (as determined by CMV antigenemia) and clinical symptoms. Recovery from CMV infection coincided with expansion of the CD8bright and CD56+ subsets and with increased expression of the activation marker HLA-DR. Primary infection was associated with activation of both subsets, whereas during secondary infection, mainly CD8bright cells responded. Progressive CMV disease (requiring antiviral treatment) and relapse occurred in association with low numbers of activated CD8bright and CD56+ cells. Lymphocyte activation and antibody responses against CMV often occurred simultaneously, but different kinetics of these responses in some patients indicated that cellular responses are necessary to control viral replication, whereas humoral responses alone may be insufficient. Monitoring of lymphocyte activation may provide clinically useful information during CMV infection.

Quantitative measurement of cytomegalovirus-specific IgG and IgM antibodies in relation to cytomegalovirus antigenaemia and disease activity in kidney recipients with an active cytomegalovirus infection.

In a longitudinal investigation 103 kidney recipients were studied with respect to the development of cytomegalovirus (CMV) specific antibodies of the IgG and IgM class, in relation to the detection of CMV antigenaemia (immediate early antigen, IEA), in weekly obtained blood samples during the first 3 months after transplantation. In 15 out of 49 (31%) seronegative patients a primary infection occurred, which was characterized by a quick rise in IgM antibody followed by a slower production of IgG antibody, high maximum numbers of IEA+ cells, and a CMV syndrome in 11 patients. In 35 out of 54 (65%) seropositive patients a secondary infection occurred. After a post-operative fall in the IgG antibody, which was also found in patients without an active infection and which was accompanied by a similar drop in serum albumin and IgG, a second dip in IgG antibody was found 6 days before the first IEA+ leucocyte appeared in the blood. This was followed by a significant increase, indicative of an active immune response in consequence of the infection, 18 days later. In 31 of these 35 patients an IgM response was found. This could be ascribed to the presence of rheumatoid factor activity in 20 of them. Eight patients who showed a transient rise in IgG antibody between the two dips could be distinguished from the remaining ones by a lower maximum number of IEA+ cells and less severe disease symptoms. The described results suggest that (i) an adequate humoral immune system may prevent symptomatic CMV disease in secondary infections; and (ii) CMV-specific antibodies may be removed from the circulation by antigens present in infected tissues before CMV antigenaemia becomes detectable.

Isolation of highly purified lymphocyte subsets for functional studies by means of an indirect rosette technique.

An indirect rosette assay, utilizing ox erythrocytes (RBC) coupled with rabbit anti-mouse IgG and lymphocytes sensitized with monoclonal mouse antibodies against membrane markers, was used for purification of lymphocyte subsets that were functionally intact. Either peripheral blood mononuclear cells (PBMC) or T lymphocytes isolated by sheep RBC rosetting could be used as starting material for obtaining pure T-cell subsets (T4 or T8). The following steps of the method were evaluated: the procedure of coupling rabbit anti-mouse IgG to ox RBC via the CrCl3 method, the experimental conditions for specific rosetting, and the use of Percoll for the separation of rosettes from the non-rosetting cells. Under optimal experimental conditions the recovery of positively selected cells was 45-55% of the cells originally present in the PBMC. The purity of these cells reached a value of more than 95%, whereas the contamination of the depleted fraction was less than 3%. The functional integrity, manifesting itself as proliferation after mitogen stimulation and as regulatory influences on in vitro Ig synthesis, appeared to be unimpaired. The described technique may be applied to the purification of various cell subpopulations for functional studies, provided monoclonal antibodies against membrane antigens are available.

Inhibition of binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels by local anesthetics.

The effects of several local anesthetics on the binding of ligands to receptors associated with voltage-sensitive sodium channels in rat brain synaptosomes have been examined. In the presence of 0.3 microM scorpion toxin, the 13 local anesthetics tested inhibited the specific binding of [3H]batrachotoxinin A 20 alpha-benzoate [( 3H]BTX-B), a ligand which binds to a receptor site responsible for the activation of sodium channel ion flux, in a dose-dependent fashion, with KD values ranging from 1.2 microM for tetracaine to 1.58 mM for benzocaine. A plot of log KD from these binding experiments against log K0.5 for inhibition of sodium currents by local anesthetics from electrophysiological experiments yielded a regression line with a slope of 0.84 and a correlation coefficient, r, of 0.86, demonstrating that the inhibition of [3H]BTX-B binding by local anesthetics occurs within a concentration range of physiological relevance. Tetracaine had little effect on basal 125I-labeled scorpion toxin binding to synaptosomes in the absence of batrachotoxin. However, in the presence of batrachotoxin, tetracaine inhibited the batrachotoxin-dependent increase in scorpion toxin binding (KD = 2.0 microM) in a dose-dependent manner, suggesting that inhibition of [3H]BTX-B binding by local anesthetics does not occur through binding at the scorpion toxin binding site. The inhibition of [3H]BTX-B binding by lidocaine was reversible within 30 min when samples were diluted from 10(-3)M to 10(-4) M lidocaine. Scatchard analysis of [3H]BTX-B binding to synaptosomes showed that bupivacaine and tetracaine reduced receptor affinity without decreasing maximal binding capacity. This reduction in receptor affinity in the presence of local anesthetics appears to be due, at least in part, to an increased rate of ligand dissociation from the receptor-ligand complex, suggesting an indirect allosteric mechanism for the inhibition of [3H]BTX-B binding by local anesthetics. Analysis of the effects of local anesthetics in terms of an allosteric model of drug action showed that they bind to inactive states of sodium channels with at least a 10-fold higher affinity than active states. A 7-fold difference in KD for inhibition of [3H]BTX-B binding between the local anesthetic stereoisomers RAC 109 I and RAC 109 II was observed. Similarly, the dissociation rate constant for the [3H]BTX-B/receptor complex was increased 9.3-fold in the presence of RAC 109 II and 4.3-fold in the presence of a comparable concentration of RAC 109 I.(ABSTRACT TRUNCATED AT 250 WORDS)