Characterization of a novel multifunctional resveratrol derivative for the treatment of atrial fibrillation.

BACKGROUND AND PURPOSE: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk for stroke, heart failure and cardiovascular-related mortality. Candidate targets for anti-AF drugs include a potassium channel K(v)1.5, and the ionic currents I(KACh) and late I(Na), along with increased oxidative stress and activation of NFAT-mediated gene transcription. As pharmacological management of AF is currently suboptimal, we have designed and characterized a multifunctional small molecule, compound 1 (C1), to target these ion channels and pathways. EXPERIMENTAL APPROACH: We made whole-cell patch-clamp recordings of recombinant ion channels, human atrial I(Kur), rat atrial I(KACh), cellular recordings of contractility and calcium transient measurements in tsA201 cells, human atrial samples and rat myocytes. We also used a model of inducible AF in dogs. KEY RESULTS: C1 inhibited human peak and late K(v)1.5 currents, frequency-dependently, with IC50 of 0.36 and 0.11 µmol·L(-1) respectively. C1 inhibited I(KACh)(IC50 of 1.9 µmol·L(-1)) and the Na(v)1.5 sodium channel current (IC50s of 3 and 1 µmol·L(-1) for peak and late components respectively). C1 (1 µmol·L(-1)) significantly delayed contractile and calcium dysfunction in rat ventricular myocytes treated with 3 nmol·L(-1) sea anemone toxin (ATX-II). C1 weakly inhibited the hERG channel and maintained antioxidant and NFAT-inhibitory properties comparable to the parent molecule, resveratrol. In a model of inducible AF in conscious dogs, C1 (1 mg·kg(-1)) reduced the average and total AF duration. CONCLUSION AND IMPLICATIONS: C1 behaved as a promising multifunctional small molecule targeting a number of key pathways involved in AF.

Triton X-100 inhibits L-type voltage-operated calcium channels.

Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol·L(-1) to 3 µmol·L(-1)) caused reversible concentration-dependent inhibition of recombinant L-type VOCC (CaV 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic ß-cells. In functional studies, TX-100 (165 nmol·L(-1) to 3.4 µmol·L(-1)) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 µmol·L(-1)) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic ß-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays.

Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis of insulin granules.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from ß-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking ß-cell ABCA1. RESEARCH DESIGN AND METHODS: Calcium imaging, patch clamp, and membrane capacitance were used to assess the effect of ABCA1 deficiency on calcium flux, ion channel function, and exocytosis in islet cells. Electron microscopy was used to analyze ß-cell ultrastructure. The quantity and distribution of proteins involved in insulin-granule exocytosis were also investigated. RESULTS: We show that a lack of ß-cell ABCA1 results in impaired depolarization-induced exocytotic fusion of insulin granules. We observed disturbances in membrane microdomain organization and Golgi and insulin granule morphology in ß-cells as well as elevated fasting plasma proinsulin levels in mice in the absence of ß-cell ABCA1. Acute cholesterol depletion rescued the exocytotic defect in ß-cells lacking ABCA1, indicating that elevated islet cholesterol accumulation directly impairs granule fusion and insulin secretion. CONCLUSIONS: Our data highlight a crucial role of ABCA1 and cellular cholesterol in ß-cells that is necessary for regulated insulin granule fusion events. These data suggest that abnormalities of cholesterol metabolism may contribute to the impaired ß-cell function in diabetes.

Inhibition of beta-cell sodium-calcium exchange enhances glucose-dependent elevations in cytoplasmic calcium and insulin secretion.

OBJECTIVE: The sodium-calcium exchanger isoform 1 (NCX1) regulates cytoplasmic calcium (Ca(2+)(c)) required for insulin secretion in beta-cells. NCX1 is alternatively spliced, resulting in the expression of splice variants in different tissues such as NCX1.3 and -1.7 in beta-cells. As pharmacological inhibitors of NCX1 splice variants are in development, the pharmacological profile of beta-cell NCX1.3 and -1.7 and the cellular effects of NCX1 inhibition were investigated. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to examine the pharmacological profile of the NCX1 inhibitor KB-R7943 on recombinant NCX1.3 and -1.7 activity. Ca(2+) imaging and membrane capacitance were used to assess the effects of KB-R7943 on Ca(2+)(c) and insulin secretion in mouse and human beta-cells and islets. RESULTS: NCX1.3 and -1.7 calcium extrusion (forward-mode) activity was approximately 16-fold more sensitive to KB-R7943 inhibition compared with cardiac NCX1.1 (IC(50s) = 2.9 and 2.4 vs. 43.0 micromol/l, respectively). In single mouse/human beta-cells, 1 micromol/l KB-R7943 increased insulin granule exocytosis but was without effect on alpha-cell glucagon granule exocytosis. KB-R7943 also augmented sulfonylurea and glucose-stimulated Ca(2+)(c) levels and insulin secretion in mouse and human islets, although KB-R7943 was without effect under nonstimulated conditions. CONCLUSIONS: Islet NCX1 splice variants display a markedly greater sensitivity to pharmacological inhibition than the cardiac NCX1.1 splice variant. NCX1 inhibition resulted in glucose-dependent increases in Ca(2+)(c) and insulin secretion in mouse and human islets. Thus, we identify beta-cell NCX1 splice variants as targets for the development of novel glucose-sensitive insulinotropic drugs for type 2 diabetes.

Splice variant-dependent regulation of beta-cell sodium-calcium exchange by acyl-coenzyme As.

The sodium-calcium exchanger isoform 1 (NCX1) is intimately involved in the regulation of calcium (Ca(2+)) homeostasis in many tissues including excitation-secretion coupling in pancreatic beta-cells. Our group has previously found that intracellular long-chain acyl-coenzyme As (acyl CoAs) are potent regulators of the cardiac NCX1.1 splice variant. Despite this, little is known about the biophysical properties of beta-cell NCX1 splice variants and the effects of intracellular modulators on their important physiological function in health and disease. Here, we show that the forward-mode activity of beta-cell NCX1 splice variants is differentially modulated by acyl-CoAs and is dependent both upon the intrinsic biophysical properties of the particular NCX1 splice variant as well as the side chain length and degree of saturation of the acyl-CoA moiety. Notably, saturated long-chain acyl-CoAs increased both peak and total NCX1 activity, whereas polyunsaturated long-chain acyl-CoAs did not show this effect. Furthermore, we have identified the exon within the alternative splicing region that bestows sensitivity to acyl-CoAs. We conclude that the physiologically relevant forward-mode activity of NCX1 splice variants expressed in the pancreatic beta-cell are sensitive to acyl-CoAs of different saturation and alterations in intracellular acyl-CoA levels may ultimately lead to defects in Ca(2+)-mediated exocytosis and insulin secretion.

Elevation in intracellular long-chain acyl-coenzyme A esters lead to reduced beta-cell excitability via activation of adenosine 5'-triphosphate-sensitive potassium channels.

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.

Metabolic regulation of sodium-calcium exchange by intracellular acyl CoAs.

The sodium-calcium exchanger (NCX) is a critical mediator of calcium homeostasis. In the heart, NCX1 predominantly operates in forward mode to extrude Ca(2+); however, reverse-mode NCX1 activity during ischemia/reperfusion (IR) contributes to Ca(2+) loading and electrical and contractile dysfunction. IR injury has also been associated with altered fat metabolism and accumulation of long-chain acyl CoA esters. Here, we show that acyl CoAs are novel, endogenous activators of reverse-mode NCX1 activity, exhibiting chain length and saturation dependence, with longer chain saturated acyl moieties being the most effective NCX1 activators. These results implicate dietary fat composition as a plausible determinant of IR injury. We further show that acyl CoAs may interact directly with the XIP (exchanger inhibitory peptide) sequence, a known region of anionic lipid modulation, to dynamically regulate NCX1 activity and Ca(2+) homeostasis. Additionally, our findings have broad implications for the coupling of Ca(2+) homeostasis to fat metabolism in a variety of tissues.

Acute oxygen sensing in cellular models: relevance to the physiology of pulmonary neuroepithelial and carotid bodies.

The combination of studies in native tissues and immortalised model systems during the last decade has made possible a deeper understanding of the physiology and functional morphology of arterial and airway oxygen sensors. Complementary and overlapping information from these earlier studies has allowed a detailed description of the cellular events that link decreased environmental oxygen to the release of physiologically important vasoactive transmitters. Since these basic pathways have now been defined functionally, what remains to be determined is the molecular identity of the specific proteins involved in the signal transduction pathways, and how these proteins interact to produce a full physiological response. With these goals clearly in sight, we have embarked upon a strategy that is a novel combination of proteomics and functional genomics. It is hoped this strategy will enable us to develop and refine the initial models in order to understand more completely the process of oxygen sensing in health and disease.

Airway chemotransduction: from oxygen sensor to cellular effector.

The process of sensing, transducing, and acting on environmental cues is critical to normal physiologic function. Furthermore, dysfunction of this process can lead to the development of disease. This is especially true of the homeostatic mechanisms that have evolved to maintain the carriage of O2 to respiring tissues during acute hypoxic challenge. During periods of reduced O2 availability, three major mechanisms act conjointly to increase ventilation and optimize the ventilation-perfusion ratio throughout the lung by directing pulmonary blood flow to better ventilated areas of the lung. These mechanisms are as follows: (1) increased carotid sinus nerve discharge rate to the respiratory centers of the brain, (2) intrinsic hypoxic vasoconstriction of pulmonary resistance vessels, and (3) potential local and central modulation via stimulation of neuroepithelial bodies of the lung. The key to the rapid response to the O2 signal is the ability of each of these tissues to sense acutely the changes in PO2, to transduce the signal, and for cellular effectors to initiate compensatory mechanisms that will offset rapidly the reduction in PO2 before O2 availability to tissues is compromised. This review concentrates on the signal transduction mechanism that links altered PO2 to depolarization in the recently proposed airway chemosensory element, the neuroepithelial body (and its immortalized cellular counterpart, the H146 cell line), and discusses the pertinent similarities and differences that exist between airway, carotid body, and pulmonary arteriolar O2 sensing.

Lack of contribution of mitochondrial electron transport to acute O(2) sensing in model airway chemoreceptors.

We have recently reported that the model airway chemoreceptors, H146 cells, exhibit a significant component of their oxygen-sensing transduction pathway which cannot be explained by activity of NADPH oxidase. Using patch-clamp, we have studied the transduction system linking reduced O(2) to k(+) channel inhibition and report that, in complete contrast to recent suggestions in pulmonary vasculature, O(2) sensing by the model airway chemoreceptors, H146 cells, does not require functional mitochondria. These data show, for the first time, that mitochondrial production of reactive O(2) species is not the unifying mechanism in O(2) sensing.