Low noise distributed acoustic sensor for seismology applications.

A distributed acoustic sensor (a phase optical time-domain reflectometer) configuration with a low noise level in the hertz and sub-hertz frequency ranges is proposed. The sensor scheme uses a Mach-Zehnder interferometer to generate a dual-pulse probe signal and implements the frequency stabilization of a laser source using the same interferometer as a frequency etalon. The scheme simultaneously provides a low noise level owing to the compensation of the optical path difference of interfering backscattered fields and low drift of the output signal. It has been shown experimentally that the stabilization of the laser frequency provides up to 35 dB signal/noise gain in the sub-hertz frequencies, which are of interest for seismology. The applicability of the proposed scheme is demonstrated experimentally by teleseismic earthquakes recorded by a fiber-optic cable deployed on the seabed of the Black Sea.

Random jumps in the phase-OTDR response.

In the paper, we present a qualitative analysis of the dual-pulse phase optical time domain reflectometry (phase-OTDR) response to uniform and nonuniform propagating fiber strain. It is found that on average over all realizations of scattering centers the response of the dual-pulse phase-OTDR is linear with respect to an external perturbation. Meanwhile, individual responses contain random phase jumps, which are an intrinsic property of phase-OTDR. These jumps are the result of nonlinear responses of the scattering fiber segments and arise due to interference of random backscattered fields varying in time. Two types of phase jumps are considered: π jumps and 2π jumps; the first type is caused by the fading in phase-OTDR spatial channel, while the second type occurs when a nonuniform perturbation propagates along the fiber. The origin of the phase jumps is explained by considering the simulated response on the complex plane. It is shown that the distribution of 2π jumps can be well described by the Gaussian probability mass function (PMF), provided the number of 2π jumps is large. The conducted experiments on the registration of uniform and nonuniform fiber strain confirm the presence of the jumps in the phase-OTDR response.

Distributed strain and temperature sensing over 100 km using tunable-wavelength OTDR based on MEMS filters.

The possibility of distributed wide-range strain and temperature measurements in a 100 km long optical fiber using tunable-wavelength low-coherence optical time-domain reflectometer (OTDR) is demonstrated. The specified distance range is provided by employing two narrowband microelectromechanical system (MEMS) spectral filters tuned synchronously as well as by taking advantage of Raman amplification and amplification by remotely pumped erbium-doped fiber segments built into the fiber under test. With the time of a single measurement of 10 min and the spatial resolution of about 1 m, the measurement range reached 1000 µÉ› in strain units, which is equivalent to the temperature range of 110°C.

ATP-sensitive potassium channels: metabolic sensing and cardioprotection.

The cardiovascular system operates under a wide scale of demands, ranging from conditions of rest to extreme stress. How the heart muscle matches rates of ATP production with utilization is an area of active investigation. ATP-sensitive potassium (K(ATP)) channels serve a critical role in the orchestration of myocardial energetic well-being. K(ATP) channel heteromultimers consist of inwardly-rectifying K(+) channel 6.2 and ATP-binding cassette sulfonylurea receptor 2A that translates local ATP/ADP levels, set by ATPases and phosphotransfer reactions, to the channel pore function. In cells in which the mobility of metabolites between intracellular microdomains is limited, coupling of phosphotransfer pathways with K(ATP) channels permits a high-fidelity transduction of nucleotide fluxes into changes in membrane excitability, matching energy demands with metabolic resources. This K(ATP) channel-dependent optimization of cardiac action potential duration preserves cellular energy balance at varying workloads. Mutations of K(ATP) channels result in disruption of the nucleotide signaling network and generate a stress-vulnerable phenotype with excessive susceptibility to injury, development of cardiomyopathy, and arrhythmia. Solving the mechanisms underlying the integration of K(ATP) channels into the cellular energy network will advance the understanding of endogenous cardioprotection and the development of strategies for the management of cardiovascular injury and disease progression.

Aminoglycoside-induced translational read-through in disease: overcoming nonsense mutations by pharmacogenetic therapy.

A third of inherited diseases result from premature termination codon mutations. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating human genetic disorders due to their unique ability to suppress gene translation termination induced by nonsense mutations. In preclinical and pilot clinical studies, this therapeutic approach shows promise in phenotype correction by promoting otherwise defective protein synthesis. The challenge ahead is to maximize efficacy while preventing interaction with normal protein production and function.

Bacterial enterotoxins are associated with resistance to colon cancer.

One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in under-developed countries. This geographic imbalance suggests an environmental contribution to the resistance of endemic populations to intestinal neoplasia. A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic bacteria associated with diarrheal disease. Here, a bacterial heat-stable enterotoxin was demonstrated to suppress colon cancer cell proliferation by a guanylyl cyclase C-mediated signaling cascade. The heat-stable enterotoxin suppressed proliferation by increasing intracellular cGMP, an effect mimicked by the cell-permeant analog 8-br-cGMP. The antiproliferative effects of the enterotoxin and 8-br-cGMP were reversed by L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as well as by removal of extracellular Ca(2+), or chelation of intracellular Ca(2+). In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca(2+) influx and inhibition of DNA synthesis in colon cancer cells. Induction of this previously unrecognized antiproliferative signaling pathway by bacterial enterotoxin could contribute to the resistance of endemic populations to intestinal neoplasia, and offers a paradigm for targeted prevention and therapy of primary and metastatic colorectal cancer.

Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance.

ATP-sensitive potassium (K(ATP)) channels are bifunctional multimers assembled by an ion conductor and a sulfonylurea receptor (SUR) ATPase. Sensitive to ATP/ADP, K(ATP) channels are vital metabolic sensors. However, channel regulation by competitive ATP/ADP binding would require oscillations in intracellular nucleotides incompatible with cell survival. We found that channel behavior is determined by the ATPase-driven engagement of SUR into discrete conformations. Capture of the SUR catalytic cycle in prehydrolytic states facilitated pore closure, while recruitment of posthydrolytic intermediates translated in pore opening. In the cell, channel openers stabilized posthydrolytic states promoting K(ATP) channel activation. Nucleotide exchange between intrinsic ATPase and ATP/ADP-scavenging systems defined the lifetimes of specific SUR conformations gating K(ATP) channels. Signal transduction through the catalytic module provides a paradigm for channel/enzyme operation and integrates membrane excitability with metabolic cascades.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels.

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (K(ATP)) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K(ATP) channel-dependent membrane excitability remains elusive. Here, we identify that the response of K(ATP) channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the K(ATP) channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K(ATP) channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K(ATP) channels and associated functions.

ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex.

ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.

Channelopathies of inwardly rectifying potassium channels.

Mutations in genes encoding ion channels have increasingly been identified to cause disease conditions collectively termed channelopathies. Recognizing the molecular basis of an ion channel disease has provided new opportunities for screening, early diagnosis, and therapy of such conditions. This synopsis provides an overview of progress in the identification of molecular defects in inwardly rectifying potassium (Kir) channels. Structurally and functionally distinct from other channel families, Kir channels are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. In humans, persistent hyperinsulinemic hypoglycemia of infancy, a disorder affecting the function of pancreatic beta cells, and Bartter's syndrome, characterized by hypokalemic alkalosis, hypercalciuria, increased serum aldosterone, and plasma renin activity, are the two major diseases linked so far to mutations in a Kir channel or associated protein. In addition, the weaver phenotype, a neurological disorder in mice, has also been associated with mutations in a Kir channel subtype. Further genetic linkage analysis and full understanding of the consequence that a defect in a Kir channel would have on disease pathogenesis are among the priorities in this emerging field of molecular medicine.

Pharmacological plasticity of cardiac ATP-sensitive potassium channels toward diazoxide revealed by ADP.

The pharmacological phenotype of ATP-sensitive potassium (K(ATP)) channels is defined by their tissue-specific regulatory subunit, the sulfonylurea receptor (SUR), which associates with the pore-forming channel core, Kir6.2. The potassium channel opener diazoxide has hyperglycemic and hypotensive properties that stem from its ability to open K(ATP) channels in pancreas and smooth muscle. Diazoxide is believed not to have any significant action on cardiac sarcolemmal K(ATP) channels. Yet, diazoxide can be cardioprotective in ischemia and has been found to bind to the presumed cardiac sarcolemmal K(ATP) channel-regulatory subunit, SUR2A. Here, in excised patches, diazoxide (300 microM) activated pancreatic SUR1/Kir6.2 currents and had little effect on native or recombinant cardiac SUR2A/Kir6.2 currents. However, in the presence of cytoplasmic ADP (100 microM), SUR2A/Kir6.2 channels became as sensitive to diazoxide as SUR1/Kir6. 2 channels. This effect involved specific interactions between MgADP and SUR, as it required Mg(2+), but not ATP, and was abolished by point mutations in the second nucleotide-binding domain of SUR, which impaired channel activation by MgADP. At the whole-cell level, in cardiomyocytes treated with oligomycin to block mitochondrial function, diazoxide could also activate K(ATP) currents only after cytosolic ADP had been raised by a creatine kinase inhibitor. Thus, ADP serves as a cofactor to define the responsiveness of cardiac K(ATP) channels toward diazoxide. The present demonstration of a pharmacological plasticity of K(ATP) channels identifies a mechanism for the control of channel activity in cardiac cells depending on the cellular ADP levels, which are elevated under ischemia.

Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea.

Bacteria that produce heat-stable enterotoxins (STs), a leading cause of secretory diarrhea, are a major cause of morbidity and mortality worldwide. ST stimulates guanylyl cyclase C (GCC) and accumulation of intracellular cyclic GMP ([cGMP]i), which opens the cystic fibrosis transmembrane conductance regulator (CFTR)-related chloride channel, triggering intestinal secretion. Although the signaling cascade mediating ST-induced diarrhea is well characterized, antisecretory therapy targeting this pathway has not been developed. 2-ChloroATP (2ClATP) and its cell-permeant precursor, 2-chloroadenosine (2ClAdo), disrupt ST-dependent signaling in intestinal cells. However, whether the ability to disrupt guanylyl cyclase signaling translates into effective antisecretory therapy remains untested. In this study, the efficacy of 2ClAdo to prevent ST-induced water secretion by human intestinal cells was examined. In Caco-2 human intestinal cells, ST increased [cGMP]i, induced a chloride current, and stimulated net basolateral-to-apical water secretion. This effect on chloride current and water secretion was mimicked by the cell-permeant analog of cGMP, 8-bromo-cGMP. Treatment of Caco-2 cells with 2ClAdo prevented ST-induced increases in [cGMP]i, chloride current and water secretion. Inhibition of the downstream consequences of ST-GCC interaction reflects proximal disruption of cGMP production because 8-bromo-cGMP stimulated chloride current and water secretion in 2ClAdo-treated cells. Thus, this study demonstrates that disruption of guanylyl cyclase signaling is an effective strategy for antisecretory therapy and provides the basis for developing mechanism-based treatments for enterotoxigenic diarrhea.

Evidence for direct physical association between a K+ channel (Kir6.2) and an ATP-binding cassette protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K+ channel.

Structurally unique among ion channels, ATP-sensitive K+ (KATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K+ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2deltaC37). Kir6.2deltaC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2deltaC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 exhibited single-channel activity characteristic of pancreatic KATP channels. Kir6.2deltaC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K+ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a KATP channel.

Adenosine prevents K+-induced Ca2+ loading: insight into cardioprotection during cardioplegia.

In clinical practice, hyperkalemic cardioplegia induces sarcolemmic depolarization, and therefore is used to arrest the heart during open heart operations. However, the elevated concentration of K+ that is present in cardioplegic solutions promotes intracellular Ca2+ loading, which could aggravate ventricular dysfunction after cardiac operations. This review highlights recent findings that have established, at the single cell level, the protective action of adenosine against hyperkalemia-induced Ca2+ loading. When it was added to hyperkalemic cardioplegic solutions, adenosine, at millimolar concentrations and through a direct action on ventricular cardiomyocytes, prevented K+-induced Ca2+ loading. This action of adenosine required the activation of protein kinase C, and it was effective only in cardiomyocytes with low diastolic Ca2+ levels. Of importance, adenosine did not diminish the magnitude of K+-induced membrane depolarization, allowing unimpeded cardiac arrest. Taken together, these findings provide direct support for the idea that adenosine is valuable when used as an adjunct to hyperkalemic cardioplegia. This idea has emerged from previous clinical studies that have shown improvement of the clinical outcome after cardiac operations when adenosine or related substances were used to supplement cardioplegic solutions. Further studies are required to define more precisely the mechanism of action of adenosine, and the conditions that may determine the efficacy of adenosine as a cytoprotective supplement to cardioplegia.

Ligand-insensitive state of cardiac ATP-sensitive K+ channels. Basis for channel opening.

The mechanism by which ATP-sensitive K+ (KATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac KATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. "Rundown" of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the KATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac KATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for KATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.

Operative condition-dependent response of cardiac ATP-sensitive K+ channels toward sulfonylureas.

A defining property of ATP-sensitive K+ (K[ATP]) channels is inhibition by sulfonylurea drugs, yet the response of cardiac K[ATP] channels toward sulfonylureas during myocardial ischemia is not consistent. Altered channel sensitivity toward sulfonylureas has, in part, been ascribed to antagonism by cytosolic nucleotide diphosphates, although the mechanism of interaction remains unclear. Herein, in inside-out patches excised from cardiomyocytes, we observed a dual response of K[ATP] channels toward the sulfonylurea drug, glyburide, in the presence of cytosolic UDP. Specifically, glyburide failed to inhibit spontaneous K[ATP] channel activity in the presence of UDP but inhibited UDP-induced channel activity after rundown of spontaneous channel openings. Such behavior of K[ATP] channels cannot be explained by differences in the level of channel activity or by UDP-induced displacement of glyburide. Rather, the dual response toward the sulfonylurea could be attributed to a property of K[ATP] channels to switch between operative conditions (spontaneous versus UDP-induced) each associated with a distinct responsiveness toward ligands. Conversion of post-rundown K[ATP] channels to the spontaneously operative channel condition, by Mg-ATP, restored the ability of UDP to antagonize the inhibitory action of glyburide lost after rundown, suggesting that the response of the channel to glyburide is phosphorylation dependent. The existence of distinct operative conditions of cardiac K[ATP] channels could be the basis for the inconsistent response of the channel toward sulfonylurea drugs and should be considered when sulfonylureas are used to implicate the opening of K[ATP] channels in the myocardium.

Burst kinetics of co-expressed Kir6.2/SUR1 clones: comparison of recombinant with native ATP-sensitive K+ channel behavior.

Co-expression of clones encoding Kir6.2, a K+ inward rectifier, and SUR1, a sulfonylurea receptor, reconstitutes elementary features of ATP-sensitive K+ (KATP) channels. However, the precise kinetic properties of Kir6.2/SUR1 clones remain unknown. Herein, intraburst kinetics of Kir6.2/SUR1 channel activity, heterologously co-expressed in COS cells, displayed mean closed times from 0.7 +/- 0.1 to 0.4 +/- 0.03 msec, and from 0.4 +/- 0.1 to 2.0 +/- 0.2 msec, and mean open times from 1.9 +/- 0.4 to 4.5 +/- 0.8 msec, and from 12.1 +/- 2.4 to 5.0 +/- 0.2 msec between -100 and -20 mV, and +20 to +80 mV, respectively. Burst duration for Kir6.2/SUR1 activity was 17. 9 +/- 1.8 msec with 5.6 +/- 1.5 closings per burst. Burst kinetics of the Kir6.2/SUR1 activity could be fitted by a four-state kinetic model defining transitions between one open and three closed states with forward and backward rate constants of 1905 +/- 77 and 322 +/- 27 sec-1 for intraburst, 61.8 +/- 6.6 and 23.9 +/- 5.8 sec-1 for interburst, 12.4 +/- 6.0 and 13.6 +/- 2.9 sec-1 for intercluster events, respectively. Intraburst kinetic properties of Kir6.2/SUR1 clones were essentially indistinguishable from pancreatic or cardiac KATP channel phenotypes, indicating that intraburst kinetics per se were insufficient to classify recombinant Kir6.2/SUR1 amongst native KATP channels. Yet, burst kinetic behavior of Kir6.2/SUR1 although similar to pancreatic, was different from that of cardiac KATP channels. Thus, expression of Kir6.2/SUR1 proteins away from the pancreatic micro-environment, confers the burst kinetic identity of pancreatic, but not cardiac KATP channels. This study reports the kinetic properties of Kir6.2/SUR1 clones which could serve in the further characterization of novel KATP channel clones.

The effect of some peptides from the hibernating brain on Ca2+ current in cardiac cells and on the activity of septal neurons.

The effects of the peptides TSKYR and DY isolated from the brain of hibernating ground squirrels on Ca2+ current were studied. TSKYR activated Ca2+ current in frog auricle fibers and in single cells from frog ventricle whereas DY blocked Ca2+ current in both preparations. In isolated rat and ground squirrel cardiocytes, TSKYR had no effect on Ca2+ current, and DY increased it. In brain slices of rat, DY blocked the activity of medial septal neurons. TSKYR increased activity of septal neurons at the initial phase, which was followed by decrease of neuronal activity.

Intracellular diadenosine polyphosphates: a novel family of inhibitory ligands of the ATP-sensitive K+ channel.

Intracellular diadenosine polyphosphates (Ap(n)A) are signal molecules that alert the cell under stress conditions. Herein, we review evidence that has recently identified a novel target for Ap(n)A, namely the ATP-sensitive K+ (K(ATP)) channel. These channels are abundant in pancreatic beta-cells and cardiac myocytes where they are essential in coupling the cellular metabolic state with membrane excitability. The potency and efficacy of Ap(n)A to inhibit K(ATP) channel activity were first described in cardiac K(ATP) channels, and appear similar to those of intracellular ATP, the primary ligand of K(ATP) channels. Also, the inhibitory ligand action of Ap(n)A is dependent upon the operative condition of K(ATP) channels and the presence of nucleotide diphosphates. In addition to a direct antagonism of channel opening, an indirect effect of Ap(n)A on K(ATP) channel activity has been associated with inhibition of adenylate kinase, a catalytic system believed essential for the regulation of channel opening. At present, the precise role for Ap(n)A-induced K(ATP) channel inhibition remains to be established. Yet, it is known that, under glucose challenge of pancreatic beta-cells, intracellular concentrations of Ap(n)A do increase to micromolar levels necessary to block K(ATP) channels, leading to insulin secretion. Thus, the Ap(n)A-mediated K(ATP) channel gating represents a previously unrecognized pathway of channel regulation.