Design and characteristics of a low-frequency magnetic probe for magnetic profile measurements at Wendelstein 7-X.

Equilibrium analysis in fusion devices usually relies on plasma pressure profiles and magnetic measurements outside the plasma. The kinetic profiles can give indirect information about the equilibrium magnetic field, while the stationary magnetic diagnostics cannot resolve current distributions on a smaller scale. This work presents a reciprocating magnetic probe, designed to provide direct plasma response measurements of the magnetic field in the scrape-off layer of Wendelstein 7-X. Hardware design and frequency characteristics are discussed, and a post-processing technique for extending the lower frequency cutoff of the integration scheme is presented.

A new multi-channel Mach probe measuring the radial ion flow velocity profile in the boundary plasma of the W7-X stellarator.

Ion flow velocity measurement in the edge and scraper-off layer region is beneficial to understand the confinement related phenomenon in fusion devices such as impurity transport and plays an important role in impurity control. During the Wendelstein 7-X (W7-X) operation phase 1.2a, a multi-channel (MC) Mach probe mounted on the multi-purpose manipulator has been used to measure radial profiles of edge ion flow velocity. This MC-Mach probe consists of two polar and two radial arrays of directional Langmuir pins (28 pins in total) serving for different aims, of which the polar arrays could obtain a polar distribution of ion saturation current, while the radial arrays can be used to study the dynamic process of a radially propagated event. In this paper, we report the observation of the radially outward propagation of a low frequency mode with a speed of around 200 m/s. The first measurement of the radial ion flow velocity profile using the MC-Mach probe in the boundary plasma of the W7-X with an island divertor will also be presented.

Dermoscopy diagnosis of cancerous lesions utilizing dual deep learning algorithms via visual and audio (sonification) outputs: Laboratory and prospective observational studies.

BACKGROUND: Early diagnosis of skin cancer lesions by dermoscopy, the gold standard in dermatological imaging, calls for a diagnostic upscale. The aim of the study was to improve the accuracy of dermoscopic skin cancer diagnosis through use of novel deep learning (DL) algorithms. An additional sonification-derived diagnostic layer was added to the visual classification to increase sensitivity. METHODS: Two parallel studies were conducted: a laboratory retrospective study (LABS, n = 482 biopsies) and a non-interventional prospective observational study (OBS, n = 63 biopsies). A training data set of biopsy-verified reports, normal and cancerous skin lesions (n = 3954), were used to develop a DL classifier exploring visual features (System A). The outputs of the classifier were sonified, i.e. data conversion into sound (System B). Derived sound files were analyzed by a second machine learning classifier, either as raw audio (LABS, OBS) or following conversion into spectrograms (LABS) and by image analysis and human heuristics (OBS). The OBS criteria outcomes were System A specificity and System B sensitivity as raw sounds, spectrogram areas or heuristics. FINDINGS: LABS employed dermoscopies, half benign half malignant, and compared the accuracy of Systems A and B. System A algorithm resulted in a ROC AUC of 0.976 (95% CI, 0.965-0.987). Secondary machine learning analysis of raw sound, FFT and Spectrogram ROC curves resulted in AUC's of 0.931 (95% CI 0.881-0.981), 0.90 (95% CI 0.838-0.963) and 0.988 (CI 95% 0.973-1.001), respectively. OBS analysis of raw sound dermoscopies by the secondary machine learning resulted in a ROC AUC of 0.819 (95% CI, 0.7956 to 0.8406). OBS image analysis of AUC for spectrograms displayed a ROC AUC of 0.808 (CI 95% 0.6945 To 0.9208). By applying a heuristic analysis of Systems A and B a sensitivity of 86% and specificity of 91% were derived in the clinical study. INTERPRETATION: Adding a second stage of processing, which includes a deep learning algorithm of sonification and heuristic inspection with machine learning, significantly improves diagnostic accuracy. A combined two-stage system is expected to assist clinical decisions and de-escalate the current trend of over-diagnosis of skin cancer lesions as pathological. FUND: Bostel Technologies. Trial Registration clinicaltrials.gov Identifier: NCT03362138.

Plasma impurities observed by a pulse height analysis diagnostic during the divertor campaign of the Wendelstein 7-X stellarator.

The paper reports on the optimization process of the soft X-ray pulse height analyzer installed on the Wendelstein 7-X (W7-X) stellarator. It is a 3-channel system that records X-ray spectra in the range from 0.6 to 19.6 keV. X-ray spectra, with a temporal and spatial resolution of 100 ms and 2.5 cm (depending on selected slit sizes), respectively, are line integrated along a line-of-sight that crosses near to the plasma center. In the second W7-X operation phase with a carbon test divertor unit, light impurities, e.g., carbon and oxygen, were observed as well as mid- to high-Z elements, e.g., sulfur, chlorine, chromium, manganese, iron, and nickel. In addition, X-ray lines from several tracer elements have been observed after the laser blow-off injection of different impurities, e.g., silicon, titanium, and iron, and during discharges with prefill or a gas puff of neon or argon. These measurements were achieved by optimizing light absorber-foil selection, which defines the detected energy range, and remotely controlled pinhole size, which defines photon flux. The identification of X-ray lines was confirmed by other spectroscopic diagnostics, e.g., by the High-Efficiency XUV Overview Spectrometer, HEXOS, and high-resolution X-ray imaging spectrometer, HR-XIS.

Prospects of X-ray imaging spectrometers for impurity transport: Recent results from the stellarator Wendelstein 7-X (invited).

This paper reports on the design and the performance of the recently upgraded X-ray imaging spectrometer systems, X-ray imaging crystal spectrometer and high resolution X-ray imaging spectrometer, installed at the optimized stellarator Wendelstein 7-X. High resolution spectra of highly ionized, He-like Si, Ar, Ti, and Fe as well as H-like Ar have been observed. A cross comparison of ion and electron temperature profiles derived from a spectral fit and tomographic inversion of Ar and Fe spectra shows a reasonable match with both the spectrometers. The also measured impurity density profiles of Ar and Fe have peaked densities at radial positions that are in qualitative agreement with the expectations from the He-like impurity fractional abundances, given the measured temperature profiles. Repeated measurements of impurity decay times have been demonstrated with an accuracy of 1 ms via injection of non-recycling Ti, Fe, and Mo impurities using a laser blow-off system.

Design, capabilities, and first results of the new laser blow-off system on Wendelstein 7-X.

We present a detailed overview and first results of the new laser blow-off system on the stellarator Wendelstein 7-X. The system allows impurity transport studies by the repetitive and controlled injection of different tracer ions into the plasma edge. A Nd:YAG laser is used to ablate a thin metal film, coated on a glass plate, with a repetition rate of up to 20 Hz. A remote-controlled adjustable optical system allows the variation of the laser spot diameter and enables the spot positioning to non-ablated areas on the target between laser pulses. During first experiments, clear spectral lines from higher ionization stages of the tracer ions have been observed in the X-ray to the extreme ultraviolet spectral range. The temporal behavior of the measured emission allows the estimate of transport properties, e.g., impurity transport times in the order of 100 ms. Although the strong injection of impurities is well detectable, the global plasma parameters are barely changed.

Overview of diagnostic performance and results for the first operation phase in Wendelstein 7-X (invited).

Wendelstein 7-X, a superconducting optimized stellarator built in Greifswald/Germany, started its first plasmas with the last closed flux surface (LCFS) defined by 5 uncooled graphite limiters in December 2015. At the end of the 10 weeks long experimental campaign (OP1.1) more than 20 independent diagnostic systems were in operation, allowing detailed studies of many interesting plasma phenomena. For example, fast neutral gas manometers supported by video cameras (including one fast-frame camera with frame rates of tens of kHz) as well as visible cameras with different interference filters, with field of views covering all ten half-modules of the stellarator, discovered a MARFE-like radiation zone on the inboard side of machine module 4. This structure is presumably triggered by an inadvertent plasma-wall interaction in module 4 resulting in a high impurity influx that terminates some discharges by radiation cooling. The main plasma parameters achieved in OP1.1 exceeded predicted values in discharges of a length reaching 6 s. Although OP1.1 is characterized by short pulses, many of the diagnostics are already designed for quasi-steady state operation of 30 min discharges heated at 10 MW of ECRH. An overview of diagnostic performance for OP1.1 is given, including some highlights from the physics campaigns.

Human Islets Exhibit Electrical Activity on Microelectrode Arrays (MEA).

This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.

BK channels affect glucose homeostasis and cell viability of murine pancreatic beta cells.

AIMS/HYPOTHESIS: Evidence is accumulating that Ca(2+)-regulated K(+) (K(Ca)) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K(Ca) channels for glucose homeostasis, beta cell function and viability. METHODS: Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca(2+) concentration ([Ca(2+)](c)) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay. RESULTS: BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of [Ca(2+)](c). BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (H(2)O(2), 10-100 µmol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of H(2)O(2)-induced K(+) currents revealed that BK channels contribute to the hyperpolarising K(+) current activated under conditions of oxidative stress. CONCLUSIONS/INTERPRETATION: Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus-secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress.

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells.

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.

An adenylate kinase is involved in KATP channel regulation of mouse pancreatic beta cells.

AIMS/HYPOTHESIS: In a previous study, we demonstrated that a creatine kinase (CK) modulates K(ATP) channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K(ATP) channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. METHODS: Single channel activity was measured with the patch-clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. RESULTS: The ATP sensitivity of K(ATP) channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in K(ATP) channel regulation. The observation that the stimulatory effect of AMP on K(ATP) channels was prevented by the AK inhibitor P (1),P (5)-di(adenosine-5')pentaphosphate (Ap(5)A; 20 micromol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in K(ATP) channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. CONCLUSIONS/INTERPRETATION: We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell K(ATP) channels. Thus, these enzymes determine the open probability of K(ATP) channels and eventually the actual rate of insulin secretion.

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1-/- mice.

AIMS/HYPOTHESIS: Islets or beta cells from Sur1(-/-) mice were used to determine whether changes in plasma membrane potential (V(m)) remain coupled to changes in cytosolic Ca(2+) ([Ca(2+)](i)) in the absence of K(ATP) channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca(2+)](i) influences oscillations in V(m) in sur1(-/-) beta cells. METHODS: Plasma membrane potential and ion currents were measured with microelectrodes and the patch-clamp technique. [Ca(2+)](i) was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations. RESULTS: Membrane depolarisation of Sur1(-/-) islets by arginine or increased extracellular K(+), elevated [Ca(2+)](i) and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1(-/-) islets. Oscillations in V(m) were influenced by [Ca(2+)](i) as follows: (1) elevation of extracellular Ca(2+) lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca(2+)-dependent outward current that was augmented by increased Ca(2+) influx through L-type Ca(2+) channels; (3) Ca(2+) depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1(-/-) islets, elevating [Ca(2+)](i) and insulin secretion; (4) store depletion activated a Ca(2+) influx that was not inhibitable by the L-type Ca(2+) channel blocker D600. CONCLUSIONS/INTERPRETATION: Although V(m) is largely uncoupled from glucose metabolism in the absence of K(ATP) channels, increased electrical activity leads to elevations of [Ca(2+)](i) that are sufficient to stimulate insulin secretion. In Sur1(-/-) beta cells, [Ca(2+)](i) exerts feedback mechanisms on V(m) by activating a hyperpolarising outward current and by depolarising V(m) via store-operated ion channels.

Direct interference of HIV protease inhibitors with pancreatic beta-cell function.

The aim of the present study was to evaluate whether HIV protease inhibitors directly interfere with stimulus-secretion coupling in pancreatic beta-cells. Insulin secretion was determined by a radioimmunoassay (RIA), cytosolic free Ca2+ concentration ([Ca2+]c) with the fluorescence dye fura-2 and whole-cell membrane currents with the patch-clamp technique. Glucose-induced insulin secretion was inhibited in a concentration-dependent manner by ritonavir and nelfinavir but not by indinavir. Ritonavir and nelfinavir lowered [Ca2+]c in the presence of a stimulatory glucose concentration whereas indinavir again had no effect. Ritonavir and nelfinavir completely inhibited the effect of tolbutamide, which normally increases [Ca2+]c by blocking KATP channels. This observation points to an action of both drugs on KATP channels or a step distal to these channels in stimulus-secretion coupling. Ritonavir was used to further evaluate the direct effects of HIV protease inhibitors on beta-cell ion channel currents. Unexpectedly, ritonavir inhibited neither the whole-cell KATP current nor the whole-cell L-type Ca2+ current. Tolbutamide almost completely suppressed the KATP current in the presence of ritonavir excluding that ritonavir alters the tolbutamide sensitivity of the KATP channel. Ritonavir increased the length and decreased the frequency of glucose-induced action potentials. This effect can be attributed to inhibition of voltage-dependent K+ currents. Intracellular stores seem not to be involved in the ritonavir-induced lowering of [Ca2+]c. In conclusion, different HIV protease inhibitors surprisingly reveal distinct effects on insulin secretion. Ritonavir inhibits insulin secretion by lowering [Ca2+]c but this effect is evidently independent of the opening of KATP channels or the closure of voltage-dependent Ca2+ channels, which are commonly considered to play a key role in stimulus-secretion coupling.

Oscillations of membrane potential and cytosolic Ca(2+) concentration in SUR1(-/-) beta cells.

AIMS/HYPOTHESIS: SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. METHODS: Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes. RESULTS: In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. [Ca(2+)](c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and [Ca(2+)](c) in cells from both SUR1(-/-) and SUR1(+/+) mice. CONCLUSION/INTERPRETATION: The strong dependence of Vm and [Ca(2+)](c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.

Diabetogenic effect of cyclosporin A is mediated by interference with mitochondrial function of pancreatic B-cells.

Treatment of patients after organ transplantation with the immunosuppressive drug cyclosporin A (CsA) is often accompanied by impaired glucose tolerance, thus promoting the development of diabetes mellitus. In the present article we show that 2 to 5 microM CsA diminishes glucose-induced insulin secretion of isolated mouse pancreatic islets in vitro by inhibiting glucose-stimulated oscillations of the cytoplasmic free-Ca(2+) concentration [Ca(2+)](c). This effect is not due to an inhibition of calcineurin, which mediates the immunosuppressive effect of CsA, because other calcineurin inhibitors, deltamethrin and tacrolimus, did not affect the oscillations in [Ca(2+)](c) of the B-cells. The CsA-induced decrease in [Ca(2+)](c) to basal values was not caused by a direct inhibition of L-type Ca(2+) channels. CsA is known to be a potent inhibitor of the mitochondrial permeability transition pore (PTP), which we recently suggested to be involved in the regulation of oscillations. Consequently, CsA also inhibited the oscillations of the cell membrane potential, and it is shown that these effects could not be ascribed to cellular ATP depletion. However, the mitochondrial membrane potential Delta Psi was affected by CsA by inhibiting the oscillations in Delta Psi. Interestingly, the observed reduction in [Ca(2+)](c) could be counteracted by the K(+)(ATP) channel blocker tolbutamide, indicating that the stimulus-secretion coupling was interrupted before the closure of K(+)(ATP) channels. It is concluded that CsA alters B-cell function by inhibiting the mitochondrial PTP. This terminates the oscillatory activity that is indispensable for adequate insulin secretion. Thus, CsA acts on different targets to induce the immunosuppressive and the diabetogenic effect.

Contrasting effects of alloxan on islets and single mouse pancreatic beta-cells.

Alloxan is used to induce diabetes in animals; however, the underlying mechanisms are still a matter of debate. Alloxan evoked a rapid hyperpolarization of the plasma membrane potential and suppressed electrical activity elicited by 15 mM glucose, thus terminating voltage-dependent Ca(2+) influx. Accordingly, glucose-induced oscillations in intracellular free Ca(2+) concentration were abolished. The effect of alloxan on membrane potential could not be reversed by glucose but was reversed by tolbutamide. However, the sensitivity to tolbutamide was decreased after treatment of the cells with alloxan. These effects closely resemble those described earlier for H(2)O(2). H(2)O(2) and alloxan decreased the mitochondrial membrane potential, indicating a decrease in ATP production and thus interference with cell metabolism. A decrease in ATP synthesis would explain the plasma membrane hyperpolarization observed in intact islets, reflecting the activation of ATP-dependent K(+) channels. Surprisingly, alloxan inhibited the whole-cell K(+)(ATP) current measured in single cells and the single-channel K(+)(ATP) current registered in excised patches. This inhibitory effect of alloxan is not mediated by changes in cell metabolism but seems to be due to direct interactions with the K(+)(ATP) channels via thiol-group oxidation. We have monitored the appearance of reactive oxygen species in single cells and islets treated with alloxan and H(2)O(2) for comparison. In contrast to H(2)O(2), alloxan induced the appearance of measurable reactive oxygen species only in islets but not in single cells. The results show that alloxan evokes different effects in islets and single cells, giving a possible explanation for inconsistent results reported in the past. It is concluded that alloxan exerts its diabetogenic effect by the production of H(2)O(2) in intact islets.

Dual effect of NO on K(+)(ATP) current of mouse pancreatic B-cells: stimulation by deenergizing mitochondria and inhibition by direct interaction with the channel.

Nitric oxide (NO) is assumed to contribute to the impairment of B-cell function in type 1 diabetes mellitus (IDDM). In the present paper we show that in mouse B-cells with intact metabolism authentic NO (20 microM) led to a biphasic effect on the K(+)(ATP) current, namely a transient increase and a consecutive almost complete inhibition. This resembles closely the effect that we have observed previously with the NO donor S-nitrosocysteine (SNOC, 1 mM) suggesting that merely NO caused both phases of this effect. We now demonstrate that the rise in the current amplitude was accompanied by a depolarization of the mitochondrial membrane potential DeltaPsi and a concomitant reduction in the ATP/ADP ratio. Thus, it seems likely that the increase in current amplitude is due to the interference of NO with cell metabolism. The subsequent inhibition of the K(+)(ATP) current is assumed to be caused by a direct effect on the channel since K(+)(ATP) single channel current activity measured in excised patches was strongly reduced by authentic NO and SNOC. Our data reveal new insights into the mechanisms underlying the biphasic action of NO on K(+)(ATP) channels in pancreatic B-cells.

Parallel oscillations of intracellular calcium activity and mitochondrial membrane potential in mouse pancreatic B-cells.

Insulin secretion in normal B-cells is pulsatile, a consequence of oscillations in the cell membrane potential (MP) and cytosolic calcium activity ([Ca(2+)](c)). We simultaneously monitored glucose-induced changes in [Ca(2+)](c) and in the mitochondrial membrane potential DeltaPsi, as a measure for ATP generation. Increasing the glucose concentration from 0.5 to 15 mM led to the well-known hyperpolarization of DeltaPsi and ATP-dependent lowering of [Ca(2+)](c). However, as soon as [Ca(2+)](c) rose due to the opening of voltage-dependent Ca(2+) channels, DeltaPsi depolarized and thereafter oscillations in [Ca(2+)](c) were parallel to oscillations in DeltaPsi. A depolarization or oscillations of DeltaPsi cannot be evoked by a substimulatory glucose concentration, but Ca(2+) influx provoked by 30 mM KCl was followed by a depolarization of DeltaPsi. The following feedback loop is suggested: Glucose metabolism via mitochondrial ATP production and closure of K(+)(ATP) channels induces an increase in [Ca(2+)](c). The rise in [Ca(2+)](c) in turn decreases ATP synthesis by depolarizing DeltaPsi, thus transiently terminating Ca(2+) influx.

Ca2+/calmodulin inhibition and phospholipase C-linked Ca2+ Signaling in clonal beta-cells.

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca2+ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca2+ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10-30 microM) and W-13 (30-100 microM), but not W-5 (100 microM) and W-12 (300 microM), reversibly inhibited the AVP- and bombesin-induced Ca2+ transients. As the generation of PLC-linked Ca2+ transients requires mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca2+ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K+ (45 mM)-induced Ca2+ signals (IC50, approximately 25 microM), and W-13 (100 microM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40-60%, whereas W-5 (100 microM) and W-12 (300 microM) had no effect. In addition, W-7 (100 microM) inhibited whole cell Ca2+ currents in mouse beta-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 microM), but not W-5 (30 microM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 microM) nor W-5 (30 microM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 microM) in the presence of verapamil (50 microM) was inhibited by W-7 (30 microM) but not by W-5 (30 microM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca2+ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.

Expression pattern of aquaporin water channels in the inner ear of the rat. The molecular basis for a water regulation system in the endolymphatic sac.

Mammalian aquaporins constitute a family of so far 10 related water channel proteins which mediate osmotically driven water fluxes across the plasma membrane. Because regulation of the ionic composition and osmolality of inner ear fluids is of great functional significance, we investigated the expression patterns of aquaporins in five defined areas of the rat inner ear by RT-PCR. The tissues used were stria vascularis, endolymphatic sac, Reissner's membrane, vestibulum and organ of Corti. Aquaporin 1 transcripts were detected in all tissues and are probably constitutive. Aquaporin 5 was only expressed in the organ of Corti and in Reissner's membrane. We show that aquaporin 2, so far considered to be specific to the principal cells of the renal collecting duct, is expressed in the endolymphatic sac. Aquaporin 2 expression was not detected in any other inner ear region. The postnatal appearance of aquaporin 2 transcripts in the endolymphatic sac resembled that in the kidney, i.e. it increased postnatally until day 4. The full-length DNA for aquaporin 2 was cloned from cDNA of the endolymphatic sac. It had an irrelevant Ile54Thr mutation because it could be functionally expressed in Xenopus oocytes. Also exclusively in the endolymphatic sac of the inner ear, we detected transcripts for aquaporin isoforms 3 and 4 which are known to be expressed in the renal principal cells. In the kidney, aquaporin 2 regulation involves vasopressin-stimulated, cAMP-dependent phosphorylation of Ser256 of aquaporin 2 which is stored in cytosolic vesicles. These storage vesicles also contain a serpentine calcium/polycation-sensing receptor. Vesicle shuffling to the plasma membrane involves proteins such as vesicle-associated membrane protein VAMP2, syntaxin-4 and the small GTPase Rab3a. Using RT-PCR we were able to demonstrate the expression of all of these components. By analogy the data suggest that in the endolymphatic sac of the inner ear a system for cellular water permeability is in place which may share many similarities with that characterized in the principal cells of the renal collecting duct. These findings may have a number of interesting pharmacological implications which need to be addressed in future studies.