Major Intraoperative Complications During Minimally Invasive Esophagectomy.

BACKGROUND: Studies have shown minimally invasive esophagectomy (MIE) to be a feasible surgical technique in treating esophageal carcinoma. Postoperative complications have been extensively reviewed, but literature focusing on intraoperative complications is limited. The main objective of this study was to report major intraoperative complications and 90-day mortality during MIE for cancer. METHODS: Data were collected retrospectively from 10 European esophageal surgery centers. All intention-to-treat, minimally invasive laparoscopic/thoracoscopic esophagectomies with gastric conduit reconstruction for esophageal and GE junction cancers operated on between 2003 and 2019 were reviewed. Major intraoperative complications were defined as loss of conduit, erroneous transection of vascular structures, significant injury to other organs including bowel, heart, liver or lung, splenectomy, or other major complications including intubation injuries, arrhythmia, pulmonary embolism, and myocardial infarction. RESULTS: Amongst 2862 MIE cases we identified 98 patients with 101 intraoperative complications. Vascular injuries were the most prevalent, 41 during laparoscopy and 19 during thoracoscopy, with injuries to 18 different vessels. There were 24 splenic vascular or capsular injuries, 11 requiring splenectomies. Four losses of conduit due to gastroepiploic artery injury and six bowel injuries were reported. Eight tracheobronchial lesions needed repair, and 11 patients had significant lung parenchyma injuries. There were 2 on-table deaths. Ninety-day mortality was 9.2%. CONCLUSIONS: This study offers an overview of the range of different intraoperative complications during minimally invasive esophagectomy. Mortality, especially from intrathoracic vascular injuries, appears significant.

Fit-for-Discharge Criteria after Esophagectomy: An International Expert Delphi Consensus.

There are no internationally recognized criteria available to determine preparedness for hospital discharge after esophagectomy. This study aims to achieve international consensus using Delphi methodology. The expert panel consisted of 40 esophageal surgeons spanning 16 countries and 4 continents. During a 3-round, web-based Delphi process, experts voted for discharge criteria using 5-point Likert scales. Data were analyzed using descriptive statistics. Consensus was reached if agreement was ≥75% in round 3. Consensus was achieved for the following basic criteria: nutritional requirements are met by oral intake of at least liquids with optional supplementary nutrition via jejunal feeding tube. The patient should have passed flatus and does not require oxygen during mobilization or at rest. Central venous catheters should be removed. Adequate analgesia at rest and during mobilization is achieved using both oral opioid and non-opioid analgesics. All vital signs should be normal unless abnormal preoperatively. Inflammatory parameters should be trending down and close to normal (leucocyte count ≤12G/l and C-reactive protein ≤80 mg/dl). This multinational Delphi survey represents the first expert-led process for consensus criteria to determine 'fit-for-discharge' status after esophagectomy. Results of this Delphi survey may be applied to clinical outcomes research as an objective measure of short-term recovery. Furthermore, standardized endpoints identified through this process may be used in clinical practice to guide decisions regarding patient discharge and may help to reduce the risk of premature discharge or prolonged admission.

Repeated temporary threshold shift and changes in cochlear and neural function.

A robust temporary threshold shift (TTS) can create significant primary damage to the auditory synapse, termed cochlear synaptopathy (CS). The common model applied to examination of this pathology is a single noise exposure or extended duration exposures at relatively high noise dosages. It is unclear if a single noise exposure that does not produce physiological changes consistent with CS (such as suppressed suprathreshold responses) can create evidence consistent with the pathology induced by repeated exposures. Here, we exposed 16-week (wk) old Sprague-Dawley rats to repeated noise exposures (4 consecutive days, 8-16 kHz octave-band of noise, 97 dB SPL for 2 h) and examined measures of cochlear function (distortion product otoacoustic emissions) and auditory neural integrity (auditory brainstem response, wave 1 amplitude). Our results demonstrated a mean maximal threshold shift of 16 dB at 24 h post the initial noise exposure. Subsequent daily repeated exposures (4 consecutive days) resulted in diminished threshold shift at 24 h post repeated TTS. In addition to recovered thresholds, no sustained reduction in suprathreshold responses was observed. The findings are consistent with conditioning literature suggesting diminished TTS with repeated exposures. Repeated TTS that was not individually synaptopathic did not produce physiological evidence consistent with acute CS.

Preemptive endoluminal vacuum therapy to reduce anastomotic leakage after esophagectomy: a game-changing approach?

Endoluminal vacuum therapy (EVT) is an accepted treatment for anastomotic leakage (AL) after esophagectomy. A novel concept is to use this technology in a preemptive setting, with the aim to reduce the AL rate and postoperative morbidity. Preemptive EVT (pEVT) was performed intraoperatively in 19 consecutive patients undergoing minimally invasive esophagectomy, immediately after completion of esophagogastrostomy. Twelve patients (63%) were high-risk cases with severe comorbidity. The EVT device was removed routinely three to six (median 5) days after esophagectomy. The endpoints of this study were AL rate and postoperative morbidity. There were 20 anastomoses at risk in 19 patients. One patient (5.3%) experienced major morbidity (Clavien-Dindo grade IIIb) unrelated to anastomotic healing. He underwent open reanastomosis at postoperative day 12 with pEVT for redundancy of the gastric tube and failure of transition to oral diet. Mortality after 30 days was 0% and anastomotic healing was uneventful in 19/20 anastomoses (95%). One minor contained AL healed after a second course of EVT. Except early proximal dislodgement in one patient, there were no adverse events attributable to pEVT. The median comprehensive complication index 30 days after surgery was 20.9 (IQR 0-26.2). PEVT appears to be a safe procedure that may have the potential to improve surgical outcome in patients undergoing esophagectomy.

Benefit of 18F-fluorocholine PET imaging in parathyroid surgery.

OBJECTIVES: To assess the additional diagnostic value of 18F-fluorocholine PET imaging in preoperative localization of pathologic parathyroid glands in clinically manifest hyperparathyroidism in case of negative or conflicting ultrasound and scintigraphy results. METHODS: A retrospective, single-institution study of 26 patients diagnosed with hyperparathyroidism. In cases where ultrasound and scintigraphy failed to detect the location of an adenoma in order to allow a focused surgical approach, an additional 18F-fluorocholine PET scan was performed and its results were compared with the intraoperative findings. RESULTS: A total of 26 patients underwent 18F-fluorocholine PET/CT (n = 11) or PET/MRI (n = 15). Adenomas were detected in 25 patients (96.2%). All patients underwent surgery, and the location predicted by PET hybrid imaging was confirmed intraoperatively by frozen section and adequate parathyroid hormone drop after removal. None of the patients needed revision surgery during follow-up. CONCLUSIONS: These results demonstrate that 18F-fluorocholine PET imaging is a highly accurate method to detect parathyroid adenomas even in case of previous localization failure by other imaging examinations. KEY POINTS: • With 18 F-fluorocholine PET imaging, parathyroid adenomas could be detected in 96.2%. • 18 F-fluorocholine imaging is a highly accurate method to detect parathyroid adenomas. • We encourage its use, where ultrasound fails to detect an adenoma.

Left-Sided Living Kidney Donation Leads to Transiently Reduced Adrenocortical Responsiveness.

Living kidney donation is safe and established, but can lead to long-term complications such as chronic fatigue. Since the adrenal vein is usually transected during left-sided donor nephrectomy-which is not necessary on the right-we hypothesized that venous congestion might lead to an impairment of adrenal function, offering a possible explanation. In this prospective open label, monocentric cohort study, adrenal function was compared in left- and right-sided living kidney donors. The primary endpoint was plasma cortisol response to low-dose adrenocorticotropic hormone (ACTH) stimulation. Secondary endpoints included plasma renin and ACTH concentration as well as adrenal volume in response to donor nephrectomy. A total of 30 healthy donors-20 left- and 10 right-sided donations-were included. On postoperative day 1, response to low-dose ACTH stimulation was intact, but significantly lower after left-sided donor nephrectomy. After 28 days, adrenal responsiveness to ACTH stimulation did not differ any longer. Magnetic resonance imaging volumetry showed no significant adrenal volume change over 4 weeks, neither after left- nor after right-sided nephrectomy. In conclusion, left-sided living kidney donation entails a transiently reduced adrenocortical responsiveness, which returns to baseline after 28 days.

An outcome and cost analysis of anal fistula plug insertion vs endorectal advancement flap for complex anal fistulae.

AIM: The study aimed to compare the rate of success and cost of anal fistula plug (AFP) insertion and endorectal advancement flap (ERAF) for anal fistula. METHOD: Patients receiving an AFP or ERAF for a complex single fistula tract, defined as involving more than a third of the longitudinal length of of the anal sphincter, were registered in a prospective database. A regression analysis was performed of factors predicting recurrence and contributing to cost. RESULTS: Seventy-one patients (AFP 31, ERAF 40) were analysed. Twelve (39%) recurrences occurred in the AFP and 17 (43%) in the ERAF group (P = 1.00). The median length of stay was 1.23 and 2.0 days (P < 0.001), respectively, and the mean cost of treatment was €5439 ± €2629 and €7957 ± €5905 (P = 0.021), respectively. On multivariable analysis, postoperative complications, underlying inflammatory bowel disease and fistula recurring after previous treatment were independent predictors of de novo recurrence. It also showed that length of hospital stay ≤ 1 day to be the most significant independent contributor to lower cost (P = 0.023). CONCLUSION: Anal fistula plug and ERAF were equally effective in treating fistula-in-ano, but AFP has a mean cost saving of €2518 per procedure compared with ERAF. The higher cost for ERAF is due to a longer median length of stay.

GSK3ß phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21.

KLF6, a ubiquitously expressed Krüppel-like transcription factor, is frequently inactivated in human cancer and has significant roles in cellular proliferation, apoptosis, differentiation and development. A key mechanism of KLF6-mediated growth suppression is through p53-independent transactivation of p21. Several cancer-derived KLF6 mutants lead to the loss of p21-mediated growth suppression through an unknown mechanism. Because several colorectal cancer and hepatocellular carcinoma-derived KLF6 mutations affect a glycogen synthase kinase 3ß (GSK3ß) phosphorylation consensus site, we investigated the role of GSK3ß in the regulation of KLF6 function. Based on transient transfection, GSK3ß augments the transactivation of a p21 promoter luciferase by KLF6. Reciprocal co-immunoprecipitation of hemagglutinin (HA)-GSK3ß and Flag-KLF6 validated the interaction between these two proteins. KLF6 phosphorylation is augmented in the presence of GSK3ß based on in vitro and in vivo (32)P incorporation assays. Site-directed mutagenesis of the candidate phosphorylation sites to alanines ('KLF6-4A' phosphomutant) eliminated a higher molecular weight phosphorylated isoform of KLF6 based on western blot. GSK3ß augmented the transactivation by wild-type KLF6, but not KLF6-4A, towards the p21 promoter, and increased p21 protein. Functionally, GSK3ß enhanced KLF6-mediated growth suppression, which was abrogated by the KLF6-4A phosphomutant. These data establish that GSK3ß directly phosphorylates KLF6, which augments its induction of p21 and resultant growth suppression. This interaction may account for the growth-promoting effects of cancer-derived KLF6 mutants that lack tumor suppressor activity.

WE-C-217A-03: Biology versus Epidemiology: The Need for an Integrated Model of Radiation Risk.

The lifetime attributable risk estimates from the National Academy of Sciences BEIR VII report have been used by a number of authors to estimate cancer mortality caused by radiation exposure from medical diagnostic radiology exams. This controversial practice assumes that the dose response relationship between radiation and cancer is linear with no threshold (LNT). For purposes of protecting public health, use of the LNT model is widely accepted. But is it appropriate for estimating risk to individuals exposed to low doses of radiation from medical procedures? Radiation biology research demonstrates that not all biological processes are linear. Italso has provided data that support not only LNT but supra linear and sub linear response models. Results from epidemiology studies can also be used to support the use of any of these models, but the confidence intervals are much larger. Since we can't prove which model is correct, for purposes of protecting patients we assume that any exposure has the potential for harm and we use optimization to keep exposures as low as reasonably achievable.Several areas of research are contributing insight into this dilemma, but they still leave several important questions unanswered: • How can we accurately extrapolate low-dose biological effects generated in the laboratory to risk in a human? • Is extrapolation from high dose, high dose rate, acute exposures appropriate when human exposures are primarily chronic low dose exposures. Epidemiology alone is unlikely to provide information that will resolve this dilemma. The numbers of individuals required in a sample are too large, and the homogeneity among subjects is lacking. Reliance on radiation biology research alone is problematic because the research is focused primarily on mechanisms and not risk. This paper will present an overview of the issues and suggest areas of research that may contribute to our understanding of the level of risk associated with low doses of medical radiation. LEARNING OBJECTIVES: 1. List the current biological mechanisms that are affected by low doses of ionizing radiation. 2. Describe the dilemma of risk extrapolation based on current knowledge of biological effects of radiation. 3. Discuss the limitations of extrapolating lifetime attributable risk estimates to cancer mortality for low-dose medical procedures.

[Microarray technique for component resolved diagnosis (CRD) in type-I allergies. An innovative technology at the border between research tool and routine diagnostics]. / Allergen-Microarrays für die molekulare Komponentendiagnostik von Typ-I-Allergien. An der Schwelle von der Forschung zur Routinediagnostik.

Approximately 25% of the population in industrialized countries suffer from IgE-associated Type-1 allergies. Multiple allergens can be tested simultaneously in one assay by using the protein microarray. Moreover, it is possible to measure more than one analytical parameter (e.g. allergen specific IgEs and IgGs) in one assay by combining different fluorescent markers with specific secondary antibodies. The different allergen components that are of interest are immobilized on a planar surface. By adding the patient's serum (a smaller amount of serum is needed compared to an immunoassay) the inherent IgE antibodies are captured by the corresponding allergens. Secondary fluorescing anti-IgE antibodies are added subsequently, thus the intensity of each spot on the microarray can be measured by using a biochipscanner. The detected signal is then transformed into quantitative data, which allows the classification of the patient's serum IgE level for the tested allergens. There are different approaches to reduce the complexity of the original extracts used for the production of the solid microarray phase to a smaller number of relevant pathogenic molecules. The component-resolved diagnosis still needs to be clinically validated, but initial studies show positive results concerning the sensitivity and specificity of the protein microarray. Protein microarrays are promising tools for screening diagnoses in allergic diseases as well as for the improvement of allergen-specific immunotherapy.

[Cine-MRI contribution to assess swallowing mechanism and oro-pharyngeal dysphagia]. / Etude de la déglutition et des dysphagies oro-pharyngées par ciné-IRM.

OBJECTIVE: This study aimed at evaluating the performance of Cine-MRI to assess swallowing in patients previously treated for head and neck cancer. MATERIALS AND METHODS: 10 healthy control subjects and a cohort of 10 patients with 8 partial glossectomies, 1 total laryngectomy and 1 glossolaryngectomy underwent imaging from October 2005 to February 2007. The MRI examinations were performed on a 1.5 Tesla system (Siemens Avanto), with True-Fisp sequences (TR = 170 ms, TE = 1 ms, slice thickness = 10 mm) at a rate of 8 pictures per second, during dry swallowing. RESULTS: Results are relevant for real-time spatial resolution from lips to larynx and dynamic motions analyses of tongue, velum, posterior pharyngeal wall and larynx during dry swallowing. Oro-pharyngo-laryngeal occlusion deficiency induces aspiration in case of partial glossectomy. Total laryngectomy modifies tongue, velum and pharynx landmarks. CONCLUSION: Cine-MRI i) provides functional insight from the oral cavity to the larynx, ii) gives accurate informations about impairments due to the pathology and its treatment, iii) completes others investigations like fiberoptic endoscopy or transit time, iiii) allows a precise analysis of the muscular movements involved in the deficient swallowing mechanism, in order to optimize rehabilitative strategies and results.

Multicentre randomised phase III trial comparing Tamoxifen alone or with Transarterial Lipiodol Chemoembolisation for unresectable hepatocellular carcinoma in cirrhotic patients (Fédération Francophone de Cancérologie Digestive 9402).

The FFCD 9402 multicentre phase III trial was designed to compare the effects of the combination of Transarterial Lipiodol Chemoembolisation (TACE) and tamoxifen with tamoxifen alone on overall survival and quality of life in the palliative treatment of hepatocellular carcinoma with cirrhosis. From 1995 to 2002, 138 patients were randomised between the two groups. One hundred and twenty three patients were eligible including 61 in the Tamoxifen group and 62 in the TACE group. Baseline characteristics were similar: Child-Pugh class A: 70%, alcoholic cirrhosis: 76%, Okuda stage I: 71%, multinodular tumour: 70% and segmental portal vein thrombosis: 10%. At 2years, the overall survival was 22% and 25% in the Tamoxifen and TACE groups (P=.68), respectively. Multivariate analysis identified four independent prognostic factors for survival: alpha-fetoprotein (AFP)>400ng/mL (P=.008), abdominal pain (P=.011), hepatomegaly (P=.023) and Child-Pugh score (P=.032). The Spitzer Index level assessing the quality of life during follow-up did not differ between the two groups (P=.70). Amongst patients with stage Okuda I, the 2-year overall survival was 28% in the Tamoxifen group and 32% in the TACE group (P=.58). In this subgroup, two prognostic factors were statistically significant for survival: AFP>400ng/mL (P=.004) and Spitzer Index (P=.013) as shown by multivariable analysis. In conclusion, this study suggests that TACE improves neither the survival nor the quality of life in patients with HCC and cirrhosis.

Whiplash injury: cases with a long period of sick leave need biomechanical assessment.

A total of 668 cases of cervical spine disorders (CSD) sustained in automotive collisions were analysed. All cases had a minimum sick leave duration of 4 weeks. To evaluate these cases a scheme was developed that takes into account technical, medical, and biomechanical aspects. For each case, the delta-v value of the underlying collision was estimated, the medical files were analysed, and a QTF (Québec Task Force) grade was assigned. In addition, the medical history of the patient was reviewed. It was found that the QTF grade for patients with pre-existing damage of the neck or pre-existing signs differed significantly from those patients without such a history. The overall assessment, which stated the extent to which the symptoms claimed could be explained by the impact, was also found to be significantly influenced by a history of neck injury. The results of the study showed that in about 50% of the cases where the technical analysis alone would not suggest that the symptoms shown could be explained by the impact, those symptoms could be explained when patient history and the collision circumstances were taken into consideration. It also found that medical evaluation based on a QTF grade alone cannot assess the explicability of claimed CSD without taking into account the collision circumstances. Therefore, the assessment of critical individual relevant biomechanical factors is necessary.

Retreatment with interferon and ribavirin vs interferon alone according to viraemia in interferon responder-relapser hepatitis C patients: a prospective multicentre randomized controlled study.

Low pretreatment viral load has consistently been shown to be an independent predictor of sustained response (SR) in patients with chronic hepatitis C infection. We assessed the efficacy of interferon (IFN) plus ribavirin vs IFN alone in low viraemic patients (<2 millions copies/mL) who had relapsed to a previous course of IFN and the efficacy of 24 vs 48 week combination therapy in high viraemic patients. Two hundred and ninety-seven patients were randomly assigned to one of the four regimens after stratification on pretreatment viral load. All patients received IFN-alpha2b (6 million units thrice weekly for 24 weeks and 3 million units thrice weekly for 24 weeks). Patients with low viraemia received either IFN-alpha2b alone for 48 weeks (R1: 42 patients) or IFN-alpha2b plus ribavirin (600 mg/day) for 24 weeks and IFN-alpha2b alone for the next 24 weeks (R2: 48 patients). Patients with high viral load received either IFN-alpha2b plus ribavirin for 24 weeks and then IFN-alpha2b alone for the next 24 weeks (R3: 104 patients) or IFN-alpha2b plus ribavirin for 48 weeks (R4: 103 patients). In low viraemic patients the rate of SR was 37.7% in group R1 and 59.6% in group R2 (P < 0.05). In high viraemic patients, the rate of SR was 44.7% in group R3 and 51.4% in group R4 (P: NS). Thirty-one patients discontinued treatment (10.4%) without difference regarding treatment regimen. In the regimen using ribavirin we found no difference in terms of SR between patients receiving a dose of ribavirin below 10.6 mg/kg/day (55%) or over 10.6 mg/kg/day (58%). Histological improvement occurred in 70.2% of patients regardless of the regimen. Logistic regression showed that genotype 2 and 3, Knodell score <6 and alanine aminotransferase pretreatment level >3 x upper limit of normal were significantly and independently correlated with SR. In low viraemic patients who relapsed to a previous IFN treatment, combination therapy using high-dose IFN and low-dose ribavirin is better than high-dose IFN alone. In high viraemic patients there was no benefit in increasing the duration of combination therapy from 24 to 48 weeks. In this study, it was found that low dose of ribavirin can be used safely and there is no effect of ribavirin dose on SR.

Biomechanical assessment of soft tissue neck injuries in cases with long sick leave times.

A database was established by collecting 919 cases of claimed cervical spine disorders (CSDs) sustained in automotive accidents. All cases had a sick leave time of more than 4 weeks. Data was obtained from a major Swiss accident insurer. An assessment scheme was developed that took into account technical, medical, and biomechanical aspects. All cases were evaluated according to this scheme. The overall biomechanical assessment, that stated the extent to which the symptoms claimed could be explained by the impact, was found to be significantly influenced by the patient's history of CSD in terms of preexisting damage or preexisting symptoms. In 52% of the assessed cases, the CSD claimed could be explained with a combination of neck loading and also by considering the patient's medical history. Performing a solely technical analysis of the collision circumstances or a purely medical evaluation based on a Quebec Task Force (QTF) grade alone are insufficient to assess the accident-related explicability of claimed CSD. Biomechanically relevant individual factors have to be considered.

alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells.

We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both alpha9 and alpha10 subunits.

Phosphorylation of phospholamban at threonine-17 in the absence and presence of beta-adrenergic stimulation in neonatal rat cardiomyocytes.

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of Fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein.

BACKGROUND: In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. METHODS AND RESULTS: Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 21/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC(50) [mol/L] NE 5.5+/-0.1, n=12; EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC(50) [mol/L] NE 5.8+/-0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC(50) [mol/L] NE 5.7+/-0.2; EPI 5.8+/-0.1). Ventricular membranes from Fallot infants, labeled with (-)-[(125)I]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. CONCLUSIONS: Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Block of the alpha9 nicotinic receptor by ototoxic aminoglycosides.

In the present study, we report that the alpha9 nicotinic acetylcholine receptor (nAChR) expressed in Xenopus laevis oocytes is reversibly blocked by aminoglycoside antibiotics. The aminoglycosides tested blocked the alpha9 nAChR in a concentration-dependent manner with the following rank order of potency: neomycin>gentamicin>streptomycin>amikacin>kanamycin. The antagonistic effect of gentamicin was not overcome by increasing the concentration of acetylcholine (ACh), indicative of a non-competitive type of block. Blockage of ACh-evoked currents by gentamicin was found to be voltage-dependent, being more potent at hyperpolarized than at depolarized holding potentials. Furthermore, gentamicin blockage was dependent upon the extracellular Ca(2+) concentration, shown by the fact that increments in extracellular Ca(2+) significantly reduced the potency of this aminoglycoside to block the alpha9 nAChR. Possible mechanisms of blockage by the aminoglycosides are discussed. The present results suggest that the initial reversible actions of aminoglycosides at the organ of Corti, such as the elimination of the olivocochlear efferent function, are due in part to the interaction with the native alpha9-containing cholinergic receptor of the outer hair cells.