ABSTRACT
BACKGROUND AND PURPOSE: Mibefradil, a T-type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here, we have investigated the pharmacological actions of mibefradil on Orai store-operated Ca2+ channels. EXPERIMENTAL APPROACH: Human Orai1-3 cDNAs in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells with stromal interaction molecule 1 (STIM1) stable expression. The Orai currents were recorded by whole-cell and excised-membrane patch clamp. Ca2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry. KEY RESULTS: Mibefradil inhibited Orai1, Orai2, and Orai3 currents dose-dependently. The IC50 for Orai1, Orai2, and Orai3 channels was 52.6, 14.1, and 3.8 µM respectively. Outside-out patch demonstrated that perfusion of 10-µM mibefradil to the extracellular surface completely blocked Orai3 currents and single channel activity evoked by 2-APB. Intracellular application of mibefradil did not alter Orai3 channel activity. Mibefradil at higher concentrations (>50 µM) inhibited Ca2+ release but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. Inhibition on Orai channels by mibefradil was structure-related, as other T-type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or minimal effects on Orai channels. Moreover, mibefradil inhibited cell proliferation, induced apoptosis, and arrested cell cycle progression. CONCLUSIONS AND IMPLICATIONS: Mibefradil is a potent cell surface blocker of Orai channels, demonstrating a new pharmacological action of this compound in regulating cell growth and death, which could be relevant to its anti-cancer activity.
Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Mibefradil/pharmacology , ORAI1 Protein/antagonists & inhibitors , ORAI2 Protein/antagonists & inhibitors , Calcium/analysis , Calcium/metabolism , Calcium Channel Blockers/chemistry , Cell Death/drug effects , Cell Proliferation/drug effects , Cells, Cultured , HEK293 Cells , Humans , Mibefradil/chemistry , ORAI1 Protein/metabolism , ORAI2 Protein/metabolism , Optical ImagingABSTRACT
Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.
Subject(s)
Albumins/metabolism , Albuminuria/genetics , Calcium Channels/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Diabetic Nephropathies/metabolism , Epithelial Cells/metabolism , Kidney Tubules, Proximal/metabolism , ORAI1 Protein/genetics , ORAI2 Protein/genetics , Albumins/drug effects , Albuminuria/metabolism , Anilides/pharmacology , Animals , Calcium/metabolism , Calcium Channel Blockers , Calcium Channels/metabolism , Case-Control Studies , Caveolins/metabolism , Cell Line , Clathrin/metabolism , Diethylstilbestrol/pharmacology , Down-Regulation , Endocytosis , Epithelial Cells/drug effects , Estrogens, Non-Steroidal/pharmacology , Female , Humans , Hyperglycemia/genetics , Hyperglycemia/metabolism , Kidney/metabolism , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Male , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Middle Aged , Mutation , ORAI1 Protein/antagonists & inhibitors , ORAI1 Protein/metabolism , ORAI2 Protein/antagonists & inhibitors , ORAI2 Protein/metabolism , Renal Reabsorption/drug effects , Renal Reabsorption/genetics , Stromal Interaction Molecule 1/metabolism , Thiadiazoles/pharmacologyABSTRACT
Provision for end-of-life care around the world is widely variable and often poor, which leads to millions of deaths each year among people without access to essential aspects of care. However, some low- and middle-income countries have improved specific aspects of end-of-life care using innovative strategies and approaches such as international partnerships, community-based programs, and philanthropic initiatives. This article reviews the state of current global end-of-life care and examines how innovation has improved end-of-life care in Nigeria, Uganda, India, Bangladesh, Myanmar, and Jordan. Specifically, we examine how opioids have been made more available for the treatment of pain, and how training and education programs have expanded the provision of care to the dying population. Finally, we recommend actions that policy makers and individuals can take to improve end-of-life care, regardless of the income level in a country.
