[Polypharmacy among elderly patients in Luxemburg]. / Polymedikation bei älteren Patienten im Grossherzogtum Luxemburg.

BACKGROUND: Since years it was suspected that a large number and possibly inappropriate medicines were prescribed to patients > 65y. OBJECTIVE: The objective of this study was the inventory of polypharmacy and the inappropriate drugs prescribed to patients of 65 years and older during the year 2008 in Luxembourg. DESIGN: For this study anonymized data were used from the data base of the medical control administration of the social security CMSS ("administration du contrôle médical de la sécurité sociale"). RESULTS: 580 general practitioners (GPs) had written 399 401 prescriptions for 58 592 patients.1080 medical specialists had written 272 197 prescriptions for 50 609 patients. Drug therapies prescribed by GPs cost 641,26 € per patient and prescribed by medical specialists 559,12 € per patient in 2008. Quantitative polypharmacy was prescribed to 17, 91% of patients (19, 99% to women and 14, 83% to men) by GPs. Medical specialists prescribed polypharmacy to 8,65% of patients (7,98% to women and 9,59% to men) by GPs. Among the 20 most prescribed drugs, inappropriate medicines for seniors were observed in the group of GPs as well as in the group of medical specialists. Family doctors prescribed inappropriate drugs to 13, 69% of patients and medical specialists to 5,46% of patients. In both groups lorazepam, zolpidem and lormetazepam have been identified as inappropriate active pharmaceutical ingredient (API). CONCLUSION: The pivotal role of the GPs in drug therapy of the elderly could be established. Polypharmacy as well as inappropriate drug prescriptions were more frequently observed in the group of GPs than in the group of medical specialists. Benzodiazepines and analogues are the most prescribed inappropriate medications to the elderly.

Near-field and confocal surface-enhanced resonance Raman spectroscopy at cryogenic temperatures.

For laser spectroscopy at variable temperatures with high spatial resolution a combined scanning near-field optical and confocal microscope was developed. Rhodamine 6G (R6G) dye molecules dispersed on silver nano-particles or nano-clusters were investigated. For optical excitation of the molecules, either an aperture probe or a focused laser spot in confocal arrangement were employed. Raman spectra in the wavenumber range between 300 cm-1 and 3000 cm-1 at room temperatures down to 8.5 K were recorded. Many of the observed Raman lines can be associated with the structure of the adsorbed molecule. Intensity fluctuations in spectral sequences were observed down to 77 K and are indicative of single molecule sensitivity.

Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit.

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C). The interaction is restricted to the CB and IQ motifs involved in the calmodulin-mediated Ca(2+)-dependent inactivation of the DHPR, suggesting functional interactions between the two channels.

Interactions of calmodulin with two peptides derived from the c-terminal cytoplasmic domain of the Ca(v)1.2 Ca2+ channel provide evidence for a molecular switch involved in Ca2+-induced inactivation.

When opened by depolarization, L-type calcium channels are rapidly inactivated by the elevation of Ca(2+) concentration on the cytoplasmic side. Recent studies have shown that the interaction of calmodulin with the proximal part of the cytoplasmic C-terminal tail of the channel plays a prominent role in this modulation. Two motifs interacting with calmodulin in a Ca(2+)-dependent manner have been described: the IQ sequence and more recently the neighboring CB sequence. Here, using synthetic peptides and fusion proteins derived from the Ca(v)1.2 channel combined with biochemical techniques, we show that these two peptides are the only motifs of the cytoplasmic tail susceptible to interact with calmodulin. We determined the K(d) of the CB interaction with calmodulin to be 12 nm, i.e. below the K(d) of IQ-calmodulin, thereby precluding a competitive displacement of CB by IQ in the presence of Ca(2+). In place, we demonstrated that a ternary complex is formed at high Ca(2+) concentration, provided that calmodulin and the peptides are initially allowed to interact at a low Ca(2+) concentration. These results provide evidence that CB and IQ motifs interacting together with calmodulin constitute a minimal molecular switch leading to Ca(2+)-induced inactivation. In addition, we suggest that they could also be the tethering site of calmodulin.

Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain.

In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Ca(v)1.1) and RyR(1), whereas in the heart the signal is carried from the cardiac-type DHPR (Ca(v)1.2) to RyR(2) by calcium ions acting as an activator. Subtypes of both channels are expressed in the central nervous system, but their functions and mechanisms of coupling are still poorly understood. We show here that complexes immunoprecipitated from solubilized rat brain membranes with antibodies against DHPR of the Ca(v)1.2 or Ca(v)1.3 subtypes contain RyR. Only type-1 RyR is co-precipitated, although the major brain isoform is RyR(2). This suggests that, in neurons, DHPRs could communicate with RyRs by way of a strong molecular interaction and, more generally, that the physical link between DHPR and RyR shown to exist in skeletal muscle can be extended to other tissues.

Distinct kinetics of cloned T-type Ca2 + channels lead to differential Ca2 + entry and frequency-dependence during mock action potentials.

Voltage-dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low-voltage-activated T-type Ca2 + channels directly affect the membrane potential and control a number of secondary Ca2 + -dependent permeabilities. We have studied the ability of the cloned T-type channels (alpha1G,H,I) to carry Ca2 + currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T-type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 + entry saturates at different spike durations according to the channel type: 4 ms for alpha1G; 7 ms for alpha1H; and > 10 ms for alpha1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the alpha1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage-independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of alpha1G and alpha1H channels to respond to high frequency stimulation ( > 20 Hz). In contrast, the slow inactivation of alpha1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of alpha1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 + signalling.

Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney.

ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K. The cDNA encodes a 77 kDa protein presenting 62% similarity with human ClC-Kb. The protein is monoglycosylated and is expressed primarily in the Xenopus kidney. It is localized in the basolateral membranes of proximal convoluted tubules of the nephron and in the apical region of the diluting segments. Heterologous expression of xClC-K in HEK-293 cells showed that the full-length protein is glycosylated and targeted to the cell membrane, but no associated Cl- current could be observed with the patch-clamp recording technique. N-glycosylation of both the native kidney channel and the recombinant protein expressed in HEK-293 conferred on them anomalous behaviour in denaturing PAGE, which is indicative of strong interactions at the extracellular side of the plasma membrane. The expression of ClC-K channels in both mesonephric and metanephric kidneys will permit further comparative physiological studies of Cl- permeabilities at the molecular level.

Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons.

Recently, two members of a new family of Ca2+ channel alpha1 subunits, alpha1G (or CavT.1) and alpha1H (or CavT.2), have been cloned and expressed. These alpha1 subunits generate Ba2+ currents similar to the T-type Ca2+ currents present in sensory neurons. Here, we use three methods to investigate whether the T currents of nodosus ganglion neurons are encoded by members of the CavT family. PCR detected the presence of mRNA encoding both alpha1G and alpha1H, as well as a third highly related sequence, alpha1I. In situ hybridizations performed on nodosus ganglia demonstrate a high expression of alpha1H subunit RNAs. Transfection of nodosus ganglion neurons with a generic antisense oligonucleotide against this new alpha1 subunit family selectively suppresses the low-voltage-activated Ca2+ current. The antisense oligonucleotide effect increased with time after transfection and reached a maximum 3 d after treatment, indicating a 2-3 d turnover for the alpha1 proteins. Taken together, these results suggest that the T-type current present in the sensory neurons is mainly attributable to alpha1H channels. In addition, taking advantage of the high specificity of the antisense ON to the cloned channels, we showed that T-type currents greatly slowed the repolarization occurring during an action potential and were responsible for up to 51% of the Ca2+ entry during spikes. Therefore, the antisense strategy clearly demonstrates the role of low-voltage-activated Ca2+ current in affecting the afterpotential properties and influencing the cell excitability. Such tools should be beneficial to further studies investigating physiological roles of T-type Ca2+ currents.

Tests of a linear model of visual-vestibular interaction using the technique of parameter estimation.

The goal of this study was to test whether a superposition model of smooth-pursuit and vestibuloocular reflex (VOR) eye movements could account for the stability of gaze that subjects show as they view a stationary target, during head rotations at frequencies that correspond to natural movements. Horizontal smooth-pursuit and the VOR were tested using sinusoidal stimuli with frequencies in the range 1.0-3.5 Hz. During head rotation, subjects viewed a stationary target either directly or through an optical device that required eye movements to be approximately twice the amplitude of head movements in order to maintain foveal vision of the target. The gain of compensatory eye movements during viewing through the optical device was generally greater than during direct viewing or during attempted fixation of the remembered target location in darkness. This suggests that visual factors influence the response, even at high frequencies of head rotation. During viewing through the optical device, the gain of compensatory eye movements declined as a function of the frequency of head rotation (P < 0.001) but, at any particular frequency, there was no correlation with peak head velocity 9P > 0.23), peak head acceleration (P > 0.22) or retinal slip speed (P > 0.22). The optimal values of parameters of smooth-pursuit and VOR components of a simple superposition model were estimated in the frequency domain, using the measured responses during head rotation, as each subject viewed the stationary target through the optical device. We then compared the model's prediction of smooth-pursuit gain and phase, at each frequency, with values obtained experimentally. Each subject's pursuit showed lower gain and greater phase lag than the model predicted. Smooth-pursuit performance did not improve significantly if the moving target was a 10 deg x 10 deg Amsler grid, or if sinusoidal oscillation of the target was superimposed on ramp motion. Further, subjects were still able to modulate the gain of compensatory eye movements during pseudo-random head perturbations, making improved predictor performance during visual-vestibular interactions unlikely. We conclude that the increase in gain of eye movements that compensate for head rotations when subjects view, rather than imagine, a stationary target cannot be adequately explained by superposition of VOR and smooth-pursuit signals. Instead, vision may affect VOR performance by determining the context of the behavior.

Activation kinetics of AMPA receptor channels reveal the number of functional agonist binding sites.

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, see Edmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.

Identifying neuronal non-L Ca2+ channels--more than stamp collecting?

The pharmacology of the majority of Ca2+ channels in the nervous system is very limited. Although attempts have been made to constrain native Ca2+ channels into the framework provided by the six pore-forming molecules cloned to date, refined biophysical analysis of Ca2+ currents, expression techniques and the use of selective toxins have helped to identify unambiguously only a limited number of Ca2+ channels. In fact, many native Ca2+ channel activities remain as 'orphans', waiting for their molecular counterparts to be defined. In this article, Janet Nooney, Régis Lambert and Anne Feltz systematically delineate the well characterized non-L Ca2+ channel activities and the missing elements in our knowledge of the Ca2+ channel family.

T-type Ca2+ current properties are not modified by Ca2+ channel beta subunit depletion in nodosus ganglion neurons.

At the molecular level, our knowledge of the low voltage-activated Ca2+ channel (T-type) has made little progress. Using an antisense strategy, we investigated the possibility that the T-type channels have a structure similar to high voltage-activated Ca2+ channels. It is assumed that high voltage-activated channels are made of at least three components: a pore forming alpha1 subunit combined with a cytoplasmic modulatory beta subunit and a primarily extracellular alpha2delta subunit. We have examined the effect of transfecting cranial primary sensory neurons with generic anti-beta antisense oligonucleotides. We show that in this cell type, blocking expression of all known beta gene products does not affect T-type current, although it greatly decreases the current amplitude of high voltage-activated channels and modifies their voltage dependence. This suggests that beta subunits are likely not constitutive of T-type Ca2+ channels in this cell type.

Resting potential of rat cerebellar granule cells during early maturation in vitro.

The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca2+ channels and a membrane potential that can respond to changes in external K+. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca2+ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from -40 mV at the first day in vitro to -60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca2+ influx through L-type and N-type voltage-activated Ca2+ channels, functional at day 0. Gross estimations of the permeabilities of Na+ and Cl- were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K+ ions seems to underlie the evolution of the resting potential with time.

Forskolin's structural analogue 1,9-dideoxyforskolin has Ca2+ channel blocker-like action in rat cerebellar granule cells.

Forskolin, routinely used as a specific activator of the cAMP pathway, is also a blocker of various ionic channels in a cAMP-independent way. We investigated, in rat cerebellar granule cells in culture, the effects of forskolin and its structural analogue 1,9-dideoxyforskolin on Ca2+ entry. Changes in cytosolic free Ca2+ concentration ([Ca]i) were monitored using fura-2 microfluorimetry. The increase in [Ca]i observed in response to membrane depolarization by 30 mM KCI was reduced by 20% in the presence of 100 microM forskolin, and by 71% with the same concentration of 1,9-dideoxyforskolin. A dose-response curve for 1,9-dideoxyforskolin gave an estimated IC50 of 54 microM. Additional experiments using the patch-clamp technique showed that 100 microM 1,9-dideoxyforskolin inhibit voltage-activated Ca2+ currents by 63%, although forskolin had no significant effect in the same conditions. This blocking effect of 1,9-dideoxyforskolin is not specific of a given Ca2+ channel type.

Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture: probing Ca2+ channel structure and function with antisense oligonucleotides.

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Voltage-activated ionic currents in differentiating rat cerebellar granule neurons cultured from the external germinal layer.

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCl). Resting potential was shown to be -10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCl but only -28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite IK consisting in a sustained K+ current blocked by tetraethylammonium (IK(TEA)), plus a rapidly activating and inactivating TEA-insensitive IK(A). Both currents increased with time in all conditions, but after 5 days IK(A) became dominant in terms of density. IK(TEA) is likely an IK(Ca), since it was blocked by 67% in 1 mM TEA. On DIV 0, INa and ICa were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, ICa mean amplitude and current density measured at -10 mV in control condition on DIV 1 was significantly larger than those recorded in bFGF and 25 mM KCl. The order of appearance of the ionic currents, IK, ICa, and INa, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on IK(A), which increased during the first 6 days of development but could be differentially regulated by bFGF.

Maintained L-type Ca2+ channel activity in excised patches of PTX-treated granule cells of the cerebellum.

Activity of high-threshold voltage activated neuronal Ca2+ channels, including dihydropyridine-sensitive (L-type) channels, rapidly disappears during cell dialysis in whole-cell recording conditions or after excision of a patch. To date, this phenomenom has been mainly related to phosphatase or protease activity. On the other hand, it has been suggested that Ca2+ channels may be regulated by G-proteins. Therefore, disruption of this regulatory pathway may also be involved directly or indirectly in the rundown process. Here, we show that treatment of cultured cerebellar granule cells with pertussis toxin (PTX) increases to 70% the probability for excising patches that display L-type Ca2+ channels activity in the inside-out recording configuration. Quantitative study indicates that, except a half decrease in the open probability, most features of the channel activity are retained after patch excision with minor modifications. The characteristics of the channel activity did not change with time during at least the first 9 min of the inside-out configuration. In addition, comparison of unitary currents recorded in the cell-attached, configuration on treated and nontreated cells demonstrates that the PTX treatment slows the activation kinetics of the current and increases the duration of channel openings evoked at -20 mV but not at 0 mV depolarizing potential. These data suggest that L-type Ca2+ channel activity are under a tonic regulation of a PTX-sensitive mechanism, which is implied in the run-down process.

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells.

In isolated chromaffin cells, the high-voltage-activated Ca2+ current, recorded using 5 mM Ca2+ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5'-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5'-triphosphate (ITP) could not replace ATP, whereas guanosine 5'-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl2 and the liberation of a phosphate group since the ATP analogue 5'-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5'-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg2+ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5'-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca2+ concentration (pCa 6 instead of pCa 7.7, where pCa = -log10[Ca2+]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca2+ channels when going to the inside-out patch recording configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP and G proteins affect the runup of the Ca2+ current in bovine chromaffin cells.

The Ca2+ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570 +/- 165 pA and then it increased by 49 +/- 23% (n = 19, SD) over 2 +/- 1 min in the absence of adenosine 5'-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17 +/- 2 min with 2 mM ATP (n = 8). Since adenosine 5'-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca2+ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n = 11). Also, guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(2-thiodiphosphate) (GDP[ beta s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca2+ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol.l We show that these processes are only effective over a period which parallels the runup and are not additive to it.(ABSTRACT TRUNCATED AT 250 WORDS)