Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics

BACKGROUND AND PURPOSE

Loop diuretics are widely used to inhibit the Na⁺, K⁺, 2Cl⁻ co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade.

EXPERIMENTAL APPROACH

Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR.

KEY RESULTS

When added to the intracellular solution, loop diuretics inhibited CFTR Cl⁻ currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl⁻ channels with ‘very fast’ speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with ‘intermediate’ speed and drug-induced blocking events were distinct from channel closures.

CONCLUSIONS AND IMPLICATIONS

Structure–activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.     

Release of growth hormone from purified somatotrophs: role of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate.

Studies were carried out to simultaneously measure cAMP and cGMP accumulation and GH release from acutely dispersed purified somatotrophs obtained from rat adenohypophyses. cAMP accumulation was dramatically increased by both prostaglandin E2 (10(-6) M) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor, 0.5 mM) within 1 min of their addition, while there was a delay of 8--16 min before a significant increase in GH release was seen. SRIF (100, 10, or 1 ng/ml) completely blocked the stimulated release of GH. SRIF also consistently decreased the elevation of cAMP induced by the two secretagogues, but this decrease was small and not always significant. cGMP was unmeasurable (less than 0.02 fmol/1000 cells) in all of our experiments, while basal cAMP levels were about 1 fmol/1000 cells. We conclude that cAMP plays a role in the intracellular mechanisms governing GH release and that SRIF primarily acts subsequent to cAMP elevation, with a possible secondard or minor action on cAMP formation.     

Adenosine-Mediated Elevation of Cyclic 3′:5′-Adenosine Monophosphate Concentrations in Cultured Mouse Neuroblastoma Cells

Adenosine causes an increase in the concentration of cyclic AMP in mouse neuroblastoma cells. The amount of increase observed in intracellular cyclic AMP levels due to exogenous adenosine depends greatly on the concentration of a specific cyclic AMP phosphodiesterase inhibitor, 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone. Unstimulated concentrations of cyclic AMP were 29-40 pmol/mg of protein, and concentrations after addition of 0.2 mM adenosine were usually twice as high. The presence of 0.7 mM inhibitor along with 0.2 mM adenosine caused an increase in cyclic AMP levels up to 1000-2000 pmol/mg of protein. In the presence of 0.7 mM inhibitor, 2 muM adenosine gives a half-maximal cyclic AMP elevation. Theophylline blocked the elevation of cyclic AMP concentrations caused by exogenous adenosine. The data show that the cyclic AMP system of mouse neuroblastoma has the necessary receptor components to respond positively to exogenous adenosine. The results presented support a direct effect of adenosine, mediated through its control of intracellular levels, on neuronal elements of the nervous system.     

Safety and immunogenicity of a canarypox-vectored human immunodeficiency virus Type 1 vaccine with or without gp120: a phase 2 study in higher- and lower-risk volunteers

Live attenuated viral vectors that express human immunodeficiency virus (HIV) antigens are being developed as potential vaccines to prevent HIV infection. The first phase 2 trial with a canarypox vector (vCP205, which expresses gp120, p55, and protease) was conducted in 435 volunteers with and without gp120 boosting, to expand the safety database and to compare the immunogenicity of the vector in volunteers who were at higher risk with that in volunteers at lower risk for HIV infection. Neutralizing antibodies to the MN strain were stimulated in 94% of volunteers given vCP205 plus gp120 and in 56% of volunteers given vCP205 alone. CD8(+) cytotoxic T lymphocyte cells developed at some time point in 33% of volunteers given vCP205, with or without gp120. Phase 3 field trials with these or similar vaccines are needed, to determine whether efficacy in preventing HIV infection or in slowing disease progression among vaccinees who become infected is associated with the level and types of immune responses that were induced by the vaccines in this study.     

The adrenal psoas sign: surgical outcomes following a simple technique to maximize removal of extracortical adrenal tissue during bilateral laparoscopic adrenalectomy

Background

Bilateral laparoscopic adrenalectomy (BLA) is an effective therapy for the management of persistent hypercortisolism in patients after failed transphenoidal pituitary tumor resection for Cushing’s disease. Extracortical adrenal tissue has been identified as a source of persistent hypercortisolism and, if not resected along with both adrenal glands, may lead to treatment failure. We report a reliable and reproducible technique called the “psoas sign” for BLA in patients with Cushing’s disease which reduces the likelihood of retained extra-adrenal cortical rests and may reduce intraoperative complications.

Methods

A 16-year retrospective review of all consecutive patients who underwent transabdominal BLA at a single tertiary care center was performed. All patients underwent BLA utilizing the psoas sign technique and all procedures were performed replicating these predetermined surgical steps: (1) Identification of the inferior pole of the gland. (2) Identification of the inferior aspect of the adreno-caval groove on the right or the adrenal vein/renal vein confluence on the left. (3) Division of the adrenal vein. (4) Dissection and removal of the adrenal gland with clearance of all retroperitoneal fat overlying the psoas muscle.

Results

Between October 1996 and December 2012, 92 patients underwent BLA for refractory Cushing’s disease. Patients were predominantly female (90 %) with a median age of 40 years (17–71). There were 3 intraoperative complications (3.2 %), 2 conversions (2.2 %), and 1 death (1.09 %). Four patients were identified as having extracortical rests of adrenal tissue within the retroperitoneal fat (4.3 %). Mean operative time was 272 min (±79.25, n = 68) and median estimated blood loss was 50 mL (10–800 mL).

Conclusions

The psoas sign technique provides a clear view of the adrenal fossa and facilitates careful dissection of the anatomic planes around the adrenal gland. This technique is feasible, reproducible and in our experience allows for safe removal of both adrenal glands and all surrounding extracortical adrenal tissue.     

Medical tourism and bariatric surgery: who pays?

Introduction

The objective of this study was to determine the short-term cost impact that medical tourism for bariatric surgery has on a public healthcare system. Due to long wait times for bariatric surgery services, Canadians are venturing to private clinics in other provinces/countries. Postoperative care in this population not only burdens the provincial health system with intervention costs required for complicated patients, but may also impact resources allotted to patients in the public clinic.

Methods

A chart review was performed from January 2009 to June 2013, which identified 62 medical tourists requiring costly interventions related to bariatric surgery. Secondarily, a survey was conducted to estimate the frequency of bariatric medical tourists presenting to general surgeons in Alberta, necessary interventions, and associated costs. A threshold analysis was used to compare costs of medical tourism to those from our institution.

Results

A conservative cost estimate of $1.8 million CAD was calculated for all interventions in 62 medical tourists. The survey established that 25 Albertan general surgeons consulted 59 medical tourists per year: a cost of approximately $1 million CAD. Medical tourism was calculated to require a complication rate ≤28 % (average intervention cost of $37,000 per patient) to equate the cost of locally conducted surgery: a rate less than the current supported evidence. Conducting 250 primary bariatric surgeries in Alberta is approximately $1.9 million less than the modeled cost of treating 250 medical tourists returning to Alberta.

Conclusions

Medical tourism has a substantial impact on healthcare costs in Alberta. When compared to bariatric medical tourists, the complication rate for locally conducted surgery is less, and the cost of managing the complications is also much less. Therefore, we conclude that it is a better use of resources to conduct bariatric surgery for Albertan residents in Alberta than to fund patients to seek surgery out of province/country.     

Higher spatial resolution improves the interpretation of the extent of ventricular trabeculation

The ventricular walls of the human heart comprise an outer compact layer and an inner trabecular layer. In the context of an increased pre‐test probability, diagnosis left ventricular noncompaction cardiomyopathy is given when the left ventricle is excessively trabeculated in volume (trabecular vol >25% of total LV wall volume) or thickness (trabecular/compact (T/C) >2.3). Here, we investigated whether higher spatial resolution affects the detection of trabeculation and thus the assessment of normal and excessively trabeculated wall morphology. First, we screened left ventricles in 1112 post‐natal autopsy hearts. We identified five excessively trabeculated hearts and this low prevalence of excessive trabeculation is in agreement with pathology reports but contrasts the prevalence of approximately 10% of the population found by in vivo non‐invasive imaging. Using macroscopy, histology and low‐ and high‐resolution MRI, the five excessively trabeculated hearts were compared with six normal hearts and seven abnormally trabeculated and excessive trabeculation‐negative hearts. Some abnormally trabeculated hearts could be considered excessively trabeculated macroscopically because of a trabecular outflow or an excessive number of trabeculations, but they were excessive trabeculation‐negative when assessed with MRI‐based measurements (T/C <2.3 and vol <25%). The number of detected trabeculations and T/C ratio were positively correlated with higher spatial resolution. Using measurements on high resolution MRI and with histological validation, we could not replicate the correlation between trabeculations of the left and right ventricle that has been previously reported. In conclusion, higher spatial resolution may affect the sensitivity of diagnostic measurements and in addition could allow for novel measurements such as counting of trabeculations.     

Neural membrane glycoproteins associated with chicken Thy-1: an anti-idiotypic antibody study

In order to investigate the possible binding of Thy-1 to other neuronal cell surface proteins, anti-idiotypic antibodies were raised using a panel of anti-Thy-1 monoclonal antibodies. Anti-idiotypic antibodies were selected for their ability to bind to day-old chick brain membrane components in enzyme-linked immunosorbent assays (ELISA), and to bind to membrane glycoproteins as determined by Western transfer immunoblotting assays. The 5 monoclonal anti-idiotypic antibodies bind to a membrane glycoprotein component of 70 kDa, and one of the antibodies also binds to 3 higher molecular weight components of 160 kDa, 120 kDa and 90 kDa. These antibodies bind to areas of the chicken cerebellum known to be rich in Thy-1. It is postulated that these molecules are associated with Thy-1, and that the role of Thy-1 on the neuronal cell surface, may be to form complexes with, and/or to stabilise these higher molecular weight glycoproteins during synaptic development.