The Burden of Leadership: a Survey of Burnout Experiences Among Psychiatry Program Directors.

OBJECTIVE: The objectives of this study were to examine the prevalence of burnout, specify contributors to and protective factors against burnout, and gather suggestions to improve well-being in psychiatry Program Directors. METHODS: A survey regarding burnout and wellness was distributed to psychiatric Program Directors through the email listserv of the American Association of Directors of Psychiatric Residency Training (AADPRT). RESULTS: The survey response rate was 273 responses out of 880 members surveyed (31%). The majority of respondents were current residency or fellowship Program Directors or Associate Program Directors or had another current educational role (93%, 227/245). Almost half of current Program Directors or Associate Program Directors reported feeling burned out almost daily or once a week (44%, 93/210). These Program Directors reported a desire to resign (77%), experienced discrimination within the past 5 years (66%), and struggled with finding meaning in their job (44%). The most frequently endorsed contributors to burnout were increasing administrative burden and insufficient support. CONCLUSIONS: The survey findings confirm that burnout characteristics are common among respondents, associated with a desire to resign and a struggle to find meaning in the highly demanding position of Program Director or Associate Program Director. Advocacy for resources, decreased administrative overload, and increased protected time would enhance well-being in Program Directors. Most striking was the frequency of discrimination reported and its relationship to burnout. Departments may benefit from a careful review of policies, procedures, and training to decrease hostile workplaces for women, international medical graduate, and under-represented in medicine Program Directors.

Metabolic Syndrome and Salt-Sensitive Hypertension in Polygenic Obese TALLYHO/JngJ Mice: Role of Na/K-ATPase Signaling.

We have demonstrated that Na/K-ATPase acts as a receptor for reactive oxygen species (ROS), regulating renal Na+ handling and blood pressure. TALLYHO/JngJ (TH) mice are believed to mimic the state of obesity in humans with a polygenic background of type 2 diabetes. This present work is to investigate the role of Na/K-ATPase signaling in TH mice, focusing on susceptibility to hypertension due to chronic excess salt ingestion. Age-matched male TH and the control C57BL/6J (B6) mice were fed either normal diet or high salt diet (HS: 2, 4, and 8% NaCl) to construct the renal function curve. Na/K-ATPase signaling including c-Src and ERK1/2 phosphorylation, as well as protein carbonylation (a commonly used marker for enhanced ROS production), were assessed in the kidney cortex tissues by Western blot. Urinary and plasma Na+ levels were measured by flame photometry. When compared to B6 mice, TH mice developed salt-sensitive hypertension and responded to a high salt diet with a significant rise in systolic blood pressure indicative of a blunted pressure-natriuresis relationship. These findings were evidenced by a decrease in total and fractional Na+ excretion and a right-shifted renal function curve with a reduced slope. This salt-sensitive hypertension correlated with changes in the Na/K-ATPase signaling. Specifically, Na/K-ATPase signaling was not able to be stimulated by HS due to the activated baseline protein carbonylation, phosphorylation of c-Src and ERK1/2. These findings support the emerging view that Na/K-ATPase signaling contributes to metabolic disease and suggest that malfunction of the Na/K-ATPase signaling may promote the development of salt-sensitive hypertension in obesity. The increased basal level of renal Na/K-ATPase-dependent redox signaling may be responsible for the development of salt-sensitive hypertension in polygenic obese TH mice.

Prognostic Value of FDG PET/CT-Derived Parameters in Pancreatic Adenocarcinoma at Initial PET/CT Staging.

OBJECTIVE: The purpose of this study is to evaluate the performance of PET-derived parameters as prognostic markers for overall survival (OS) and progression-free survival (PFS) outcome in patients with pancreatic adenocarcinoma. MATERIALS AND METHODS: We conducted a retrospective study of 106 patients (62 men and 44 women) with histologically proven pancreatic adenocarcinoma who underwent initial staging FDG PET/CT before treatment. Peak standardized uptake value (SUV), maximum SUV (SUVmax), metabolic tumor volume, and tumor glycolytic activity of the primary pancreatic tumor were measured. Two segmentation methods were performed to obtain the metabolic tumor volume and tumor glycolytic activity for all tumors: a gradient-based segmentation model (metabolic tumor volume and tumor glycolytic activity by gradient edge detection) and a fixed-threshold model with a threshold of 50% of the lesion's SUVmax and peak SUV. Univariate and multivariate Cox regression models were developed including clinical and imaging parameters for OS and PFS. RESULTS: Multivariate Cox regression analysis showed a statistically significant association between PFS and age, SUVmax, peak SUV, and tumor glycolytic activity by gradient edge detection. There was a statistically significant difference in PFS for patients with values above and below the median cutoff points for SUVmax (hazard ratio [HR], 1.12; p < 0.01), peak SUV (HR, 1.25; p < 0.02), and tumor glycolytic activity measured by gradient edge detection (HR, 1.00; p < 0.02) of the primary tumor. However, multivariate Cox regression analysis showed a statistically significant association only between tumor glycolytic activity by gradient edge detection and OS (p = 0.04), and there was a statistically significant difference in OS between patients with values above and below the median cutoff point for the tumor glycolytic activity by gradient edge detection of the primary tumor (HR, 1.42; p = 0.05). CONCLUSION: Age, SUVmax, peak SUV, and total lesion glycolysis (i.e., tumor glycolytic activity) of the primary tumor are associated with PFS, and tumor glycolytic activity is associated with OS in patients with pancreatic adenocarcinoma.

FT-IR microspectroscopy of mouse colon tissues: insight into the chemistry of carcinogenesis and diagnostic potential.

We studied colon carcinogenesis using Fourier-transform infrared (FT-IR) microspectroscopy, an evolving method that allows the nondestructive assessment of the chemical composition of cells and tissues and of the in situ relationship between molecules, and assessed its diagnostic potential. Mid-FT-IR spectra were obtained from frozen colon tissue samples of normal (C57BL/6J) and Min (Apc(Min) mutant) mice, the latter recapitulating key features of human colon carcinogenesis. Classic spectroscopic analysis demonstrated marked differences in the Mid-FT-IR spectra between normal and dysplastic tissues, especially regarding peak positions and band intensity ratios in the regions 1800 to 985 cm(-1) and 3000 to 2700 cm(-1), reflecting changes in cellular nucleic acids, phosphates, and carbohydrates. Analysis of the spectra using the multivariate methods backpropagation neural networks, decision tree, adaboost with decision tree, and support vector machine, which interrogated the intrinsic dimensionality of IR spectra, revealed that their sensitivity was between 91.1% and 100% and their specificity between 94.1% and 100%, with the outcomes of the Support Vector Machine algorithm being identical to those of histologic analysis. FT-IR microspectroscopy holds great promise not only as a method of ascertaining changes in the chemistry of the neoplastic cells but also as a diagnostic tool, especially for early stages of carcinogenesis not detectable by other means.

Inducibility of atrial fibrillation after GP ablations and "autonomic blockade": evidence for the pathophysiological role of the nonadrenergic and noncholinergic neurotransmitters.

BACKGROUND: Recent clinical reports that used cholinergic and adrenergic blockade (CAB) as an alternative to ganglionated plexi (GP) ablation to terminate atrial fibrillation (AF) showed mixed results. We investigated the role of other neurotransmitters in AF inducibility. METHODS: In 23 pentobarbital anesthetized dogs, a left and right thoracotomy allowed the attachment of electrode catheters to the left and right pulmonary veins and atrial appendages (AA). Programmed stimulation was used to determine the effective refractory periods (ERP) and AF inducibility, measured by the window of vulnerability (WOV). AF duration in response to acetylcholine (Ach; 100 mM) applied to the AA was measured before and after GP ablation + CAB and with vagus nerve stimulation (VNS). After GP ablation + CAB, Ach induced AF duration was determined in response to vasoactive intestinal peptide (VIP) and its specific antagonist ([Ac-Tyr1,D-phe2]-VIP). RESULTS: GP ablation + CAB significantly prolonged ERP, eliminated WOV, and suppressed the duration of Ach induced AF (P ≤ 0.01 for all). Also slowing of the heart rate by VNS was essentially blocked; however, with Ach 100 mM applied to the AA, VNS, and VIP applied to the AA markedly prolonged AF duration. This effect was blocked by the VIP antagonist. CONCLUSIONS: Neither GP ablation nor CAB can fully suppress AF inducibility arising from the atrial neural network. Our findings suggest that other neurotransmitters, such as VIP released during VNS, can promote sustained AF despite GP ablation and "autonomic blockade," which may further define the substrate for AF outside the pulmonary vein-atrial junctions.

pNaKtide Inhibits Na/K-ATPase Signaling and Attenuates Obesity.

Obesity is a growing public health crisis across the world and has been recognized as an underlying risk factor for metabolic syndrome. Growing evidence demonstrates the critical role of oxidative stress in the pathophysiological mechanisms of obesity and related metabolic dysfunction. As we have established previously that Na/K-ATPase can amplify oxidative stress signaling, we aimed to explore the effect of inhibition of this pathway on obesity phenotype using the peptide antagonist, pNaKtide. The experiments performed in murine preadipocytes showed the dose-dependent effect of pNaKtide in attenuating oxidant stress and lipid accumulation. Furthermore, these in vitro findings were confirmed in C57Bl6 mice fed a high-fat diet. Interestingly, pNaKtide could significantly reduce body weight, ameliorate systemic oxidative and inflammatory milieu and improve insulin sensitivity in obese mice. Hence the study demonstrates the therapeutic utility of pNaKtide as an inhibitor of Na/K-ATPase oxidant amplification signaling to alleviate obesity and associated comorbidities.

8²Rb PET myocardial perfusion imaging is superior to 99mTc-labelled agent SPECT in patients with known or suspected coronary artery disease.

PURPOSE: We compared the quality, interpretive confidence and interreader agreement between SPECT and PET myocardial perfusion imaging (MPI) in the same group of patients. METHODS: The study group comprised 27 patients (age 55 ± 8.5 years, 12 men) with known or suspected coronary artery disease (CAD) who had undergone gated rest/stress MPI with (99m)Tc-labelled agent SPECT (with and without attenuation correction, AC), and subsequent clinical confirmation with (82)Rb PET. Three experienced readers blinded to the clinical information interpreted all MPI studies. RESULTS: Interreader agreement was significantly superior for PET studies than for SPECT studies. Following consensus interpretation, the quality of 22 % of the non-AC SPECT studies, 33 % of the AC SPECT studies and 63 % of the PET studies was assessed as excellent or good (p = 0.016). Interpretations were definitely normal or abnormal in 7 % of non-AC SPECT studies, 30 % of AC SPECT studies and 85 % of PET studies (p = 0.046). In 13 patients who had received either invasive coronary angiography or CT angiography with no significant CAD, the true-positive rate for significant CAD was higher for PET, and the true-negative rate was equal for PET and AC SPECT, and lower for non-AC SPECT. CONCLUSION: (82)Rb PET MPI, used as a confirmatory test after SPECT, offers improved image quality, interpretive confidence and interreader agreement.

Constipation--another manifestation of eosinophilic gastroenteritis.

Eosinophilic gastroenteris (EG) is a unique disease in which eosinophils penetrate into the layers of the GI tract. It is classified by the depth of the eosinophilic penetration, that can either be mucosal, muscularis or serosal. Symptoms vary with the bowel site involved as well as the depth of the eosinophilic penetration. Symptoms of the mucosal form of EG usually include nausea, vomiting, diarrhea and abdominal pain while constipation is extremely rare. We present a case of mucosal EG presenting as constipation and abdominal pain in a 43 year old female. Constipation is not a typical symptom associated with EG and while muscularis EG can cause decreased colonic motility and obstruction, constipation with mucosal EG has not been previously reported. We are presenting the first case report of constipation associated with mucosal EG. Thus EG should be considered in the differential diagnosis of patients presenting with constipation and abdominal pain and can easily be diagnosed with mucosal biopsies and treated with steroid therapy.

Atrial myxoma and bone changes: a paraneoplastic syndrome?

Atrial myxomas are the most common benign tumors of the heart and are difficult to diagnose due to a wide variety of presenting symptoms. We present a patient with a five-year history of visual loss, vertigo, ataxia, tinnitus, and bone lesions that resolved after diagnosis and resection of an atrial myxoma. This case not only highlights an unusual presentation of atrial myxomas but also raises the question of whether atrial myxomas can produce paraneoplastic syndromes, including bone abnormalities.

De Garengeot hernia masquerading as a painless groin lump: a case report.

De Garengeot's hernia is a rare subtype of femoral hernia in which the appendix is located within the herniated sac. These cases are important to report as both the diagnosis and treatment are quite challenging. We present a case of a 68-year-old gentleman with few months history of a lump in the right groin that gave him mild discomfort but no other symptoms. Initial investigations with an ultrasound did not prove to be helpful and so a plan was made to surgically explore the lump. The appendiceal tip was incarcerated within the hernial sac. The appendix was removed using an open inguinal incision with repair of the defect using a light weight partially absorbable mesh. It is important to consider the possibility of a De Garangeot's Hernia as a differential diagnosis for patients presenting with a groin lump.

Prospective Within-Patient Assessment of the Impact of an Unlabeled Octreotide Pre-dose on the Biodistribution and Tumor Uptake of 68Ga DOTATOC as Assessed by Dynamic Whole-body PET in Patients with Neuroendocrine Tumors: Implications for Diagnosis and Therapy.

PURPOSE: Gastroenteropancreatic neuroendocrine tumors (GEP NETs) are often associated with high expression of somatostatin receptors (SSTRs) which allows for PET/CT imaging with radiolabeled somatostatin analogs such as 68Ga-DOTATOC. The interplay between 68Ga-DOTATOC and the synthetic somatostatin analogs commonly used to manage patient symptoms may lead to competition between the labelled and unlabeled peptides for receptor binding sites and current product labelling recommends patients be taken off somatostatin analogs before imaging. In this study, we prospectively investigated in human patients the effect of a pre-dose of octreotide, a short-acting somatostatin analog, on the distribution of 68Ga-DOTATOC in GEP NETs and normal organs. PROCEDURE: Research participants with GEP NETs were studied on two occasions using dynamic whole-body 68Ga-DOTATOC PET/CT. The two imaging studies were performed within 21 days of each other, using an identical acquisition protocol except for the administration of 50 µg of short-acting octreotide (pre-dose) immediately before the second PET/CT. Paired t-tests were used to compare tracer uptake with and without octreotide, for tumor and various normal organs. RESULTS: Seven participants with a mean age of 53 ± 10 years were studied. Octreotide pre-dosing decreased radiotracer uptake in the normal liver and spleen by 25 % (p = 0.04) and 47 % (p = 0.05) respectively but did not significantly change uptake in tumor (p = 0.53), red marrow (p = 0.12), kidneys (p =0.57), or pituitary gland (p = 0.27). CONCLUSIONS: Our data indicate SSTR imaging can be improved with a pre-dose of unlabeled octreotide given just prior to injection of the radiotracer. These data suggest there may be no need to discontinue somatostatin analog therapy prior to PET/CT with 68Ga-DOTATOC, allowing for a simpler, less disruptive patient protocol. This approach warrants further study in a variety of settings.

Isolated perforated jejunal diverticulitis: a case report.

Jejunal diverticulosis is a rare phenomenon often identified either incidentally on imaging or intra-operatively. Complications of jejunal diverticulosis are associated with high rates of mortality. For this reason, it remains important that this pathology is considered amongst differentials for an acute abdomen. A 78-year old gentleman presented with a short history of generalized lower abdominal pain. Computer tomography scan revealed a large inflammatory abscess relating to a perforated jejunal diverticulum. The patient was taken to theatre where he underwent small bowel resection with primary anastomosis. Early cross sectional imaging is vital to allow early diagnosis and prompt management of this pathology. Small bowel resection with primary anastomosis was associated with an excellent clinical outcome.

The role of PET scanning in pancreatic cancer.

The strength of functional imaging lies in its ability to detect malignant disease irrespective of lesion morphology. In this setting, 18FDG-PET can complement management by providing a more accurate diagnosis. When combined as an adjunct to CT, 18FDG-PET can increase the sensitivity, specificity, and accuracy for detecting a pancreatic malignancy, especially in patients in whom CT alone fails to identify a discrete mass or in whom biopsy results are indeterminate. This capability is accentuated with small lesions of the pancreas. 18FDG-PET is significantly more sensitive in detecting metastatic disease than conventional CT imaging. Moreover, 18FDG-PET is able to differentiate tumor response to therapy in the postoperative setting, and could potentially serve to monitor recurrence patterns in the setting of neoadjuvant or adjuvant chemoradiotherapy. Finally, as 18FDG-PET/CT fusion modalities become more widespread and technical advances in image acquisition progress, 18FDG-PET will continue to have an increasing role in the diagnosis, staging, and surveillance of pancreatic cancer, integrating anatomic information with functional imaging.

The Na/K-ATPase α1 and c-Src form signaling complex under native condition: A crosslinking approach.

The protein-protein interactions amongst the Na/K-ATPase α1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there are arguments concerning the interaction model. The present study aims to clarify the interactions amongst the endogenous native proteins in live cells under native resting condition. Under native condition, Blue Native-PAGE and Blue Native-PAGE/SDS-PAGE 2D analyses demonstrated co-existence of the α1 subunit and c-Src in same protein complex, as well as a direct interaction between the α1 subunit and c-Src. By comparison of cleavable and non-cleavable cysteine-cysteine crosslinked samples, capillary immunoblotting analysis demonstrated that depletion of Src kinase family members (c-Src, Yes, and Fyn) or cav-1 clearly reduced the interactions of the α1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the α1 subunit. The data indicated that there are direct interactions between the α1 subunit and c-Src as well as between the α1 subunit and cav-1, but argued about the interaction between c-Src and cav-1 under the condition. Furthermore, the data also indicated the existence of different protein complexes containing the α1 subunit and c-Src, which might have different signaling functions.

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy.

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy". We describe here an experimental CKD mouse model, named 5/6th partial nephrectomy (PNx) with pole ligation, which developed uremic cardiomyopathy at four weeks post-surgery. This PNx model was performed by a two-step surgery. In step-one surgery, both poles of the left kidney were ligated. In step-two surgery, which was performed 7 days after the step-one surgery, the right kidney was removed. For the sham surgery, the same surgery procedures were performed but without pole ligation of the left kidney or removal of the right kidney. The surgical procedures are easier and less time-consuming, compared to other methods. However, the remnant functional renal mass is not as easily controlled as the renal artery ligation. Four weeks after surgery, in comparison with the sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

Protein Carbonylation of an Amino Acid Residue of the Na/K-ATPase α1 Subunit Determines Na/K-ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells.

BACKGROUND: We have demonstrated that cardiotonic steroids, such as ouabain, signaling through the Na/K-ATPase, regulate sodium reabsorption in the renal proximal tubule. By direct carbonylation modification of the Pro222 residue in the actuator (A) domain of pig Na/K-ATPase α1 subunit, reactive oxygen species are required for ouabain-stimulated Na/K-ATPase/c-Src signaling and subsequent regulation of active transepithelial (22)Na(+) transport. In the present study we sought to determine the functional role of Pro222 carbonylation in Na/K-ATPase signaling and sodium handling. METHODS AND RESULTS: Stable pig α1 knockdown LLC-PK1-originated PY-17 cells were rescued by expressing wild-type rat α1 and rat α1 with a single mutation of Pro224 (corresponding to pig Pro222) to Ala. This mutation does not affect ouabain-induced inhibition of Na/K-ATPase activity, but abolishes the effects of ouabain on Na/K-ATPase/c-Src signaling, protein carbonylation, Na/K-ATPase endocytosis, and active transepithelial (22)Na(+) transport. CONCLUSIONS: Direct carbonylation modification of Pro224 in the rat α1 subunit determines ouabain-mediated Na/K-ATPase signal transduction and subsequent regulation of renal proximal tubule sodium transport.

Tumor necrosis factor-alpha-induced changes in insulin-producing beta-cells.

The migration of macrophages and lymphocytes that produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) causes beta-cell death, leading to type 1 diabetes. Similarly, in type 2 diabetes, the adipocyte-derived cytokines including TNF-alpha are elevated in the circulation, causing inflammation and insulin resistance. Thus, the studies described in this article using TNF-alpha are relevant to furthering our understanding of the pathogenesis of diabetes mellitus. We used RINr1046-38 (RIN) insulin-producing beta-cells, which constitutively express calbindin-D(28k), to characterize the effect of TNF-alpha on apoptosis, replication, insulin release, and gene and protein expression. Western blots of TNF-alpha-treated RIN cells revealed a decrease in calbindin-D(28k). By ELISA, TNF-alpha-treated beta-cells had 47% less calbindin-D(28k) than controls. In association with the decline in calbindin-D(28k), TNF-alpha treatment of RIN cells led to a 73% greater increase in changes in intracellular calcium concentration (Delta[Ca(2+)](i)) in TNF-alpha-treated cells as compared to that in control RIN cells upon treatment with 50 mM KCl; caused a greater increase in the [Ca(2+)](i) following the addition of 5.5 microM ionomycin; increased by more than threefold the apoptotic rate, expressed as the percentage of TUNEL-positive nuclei to total nuclei; decreased the rate of cell replication by 36%; and increased and decreased selectively the expression of specific genes as determined by microarray analysis. The subcellular localizations of Bcl-2, an antiapoptotic protein, and Bax, a proapoptotic protein, within RIN cells were altered with TNF-alpha treatment such that the two were colocalized with mitochondria in the perinuclear region. We conclude that the proapoptotic action of TNF-alpha on beta-cells is manifested via decreased expression of calbindin-D(28k) and is mediated at least in part by [Ca(2+)](i).

Evaluation of osseous metastasis in bone scintigraphy.

Bone scintigraphy (BS) is an imaging tool commonly used for screening patients with cancer, especially those with high prevalence of osseous metastases including the breast, prostate, lung, thyroid, and kidney, which account for 80% of osseous metastasis. BS has been shown to be of value in the initial and subsequent treatment strategy of various malignancies. The purpose of this article is to evaluate the technical and imaging aspects of BS and to examine the present research into improved detection of osseous metastasis.

RETRACTED: pNaKtide inhibits Na/K-ATPase reactive oxygen species amplification and attenuates adipogenesis.

Obesity has become a worldwide epidemic and is a major risk factor for metabolic syndrome. Oxidative stress is known to play a role in the generation and maintenance of an obesity phenotype in both isolated adipocytes and intact animals. Because we had identified that the Na/K-ATPase can amplify oxidant signaling, we speculated that a peptide designed to inhibit this pathway, pNaKtide, might ameliorate an obesity phenotype. To test this hypothesis, we first performed studies in isolated murine preadipocytes (3T3L1 cells) and found that pNaKtide attenuated oxidant stress and lipid accumulation in a dose-dependent manner. Complementary experiments in C57Bl6 mice fed a high-fat diet corroborated our in vitro observations. Administration of pNaKtide in these mice reduced body weight gain, restored systemic redox and inflammatory milieu, and, crucially, improved insulin sensitivity. Thus, we propose that inhibition of Na/K-ATPase amplification of oxidative stress may ultimately be a novel way to combat obesity, insulin resistance, and metabolic syndrome.

Calbindin-D28k and calcium sensing receptor cooperate in MCF-7 human breast cancer cells.

The metastases of breast cancer cells to bone result in osteolysis and release of Ca2+. Ca2+ as a primary signal transducer can regulate the expression patterns of cell signaling systems. The extracellular calcium ion concentration sensing receptor CaR is a 123 kDa G-protein coupled membrane protein that resides within caveolin-rich regions as a dimer. CaR is involved in regulating several cellular processes such as proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a 28 kDa high affinity calcium-binding protein and it is involved in regulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to extracellular calcium ion concentration, [Ca2+]o, and neomycin sulfate, and spatial interactions of CaR with calbindin-D28k in MCF-7 human breast cancer cells were studied. Fura-2 loaded MCF-7 cells were exposed to increasing concentrations of CaCl2 or neomycin sulfate and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 or neomycin sulfate caused an increase in [Ca2+]i. The normalized dose response curves fitting yielded Hill co-efficient values of 4.32+/-0.63 and 1.49+/-0.14 for Ca2+ and neomycin sulfate respectively, thus indicating highly co-operative, 4-5 binding sites for Ca2+ and 1-2 binding site(s) for neomycin sulfate on CaR. The EC50 values were 21+/-1.6 mM and 43+/-3.5 micro M for CaCl2 and neomycin sulfate respectively. The confocal microscopy data, obtained by using a highly sensitive tyramide signal amplification technology for immunofluorescence detection, showed CaR and calbindin-D28k were co-localized when cells were exposed to 200 micro M neomycin sulfate, whereas in control cells there was no co-localization of these two proteins. We hypothesize that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.