Research of positioning accuracy of robot MOTOMAN SSF2000

Research of the dynamic properties, positioning precision and repeatability of MOTOMAN SSF2000 movement is presented in this article. Dynamic properties were analyzed using a mobile equipment of measuring „Machine Diagnostic Toolbox Type 9727“ with a DELL notebook. The manipulator vibrations were measured in two robot’s positions: vertical and horizontal, and at three different speeds. The research was made under three different conditions, changing the tool mass and spee

Research of positioning accuracy of robot MOTOMAN SSF2000

Research of the dynamic properties, positioning precision and repeatability of MOTOMAN SSF2000 movement is presented in this article. Dynamic properties were analyzed using a mobile equipment of measuring „Machine Diagnostic Toolbox Type 9727“ with a DELL notebook. The manipulator vibrations were measured in two robot’s positions: vertical and horizontal, and at three different speeds. The research was made under three different conditions, changing the tool mass and spee

Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome

Mutations in potassium and calcium channel genes have been associated with cardiac arrhythmias. Here, Jensen et al. show that an anion transporter chloride-bicarbonate exchanger AE3 is also responsible for the genetically-induced mechanism of cardiac arrhythmia, suggesting new therapeutic targets for this diseas

Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors

Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling

Conserved expression and functions of PDE4 in rodent and human heart

PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation–contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has ‘global’ effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts

Cyclic Nucleotide Phosphodiesterases and Compartmentation in Normal and Diseased Heart

International audienceCyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cAMP and cGMP, thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families which are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP controlling specific cell functions in response to various neurohormonal stimuli. In myocardium, the PDE3 and PDE4 families are predominant to degrade cAMP and thereby regulate cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in human, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important to degrade cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. However, these drugs do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence that these PDEs as well as other PDE families including PDE1, PDE2 and PDE9 may play important roles in cardiac diseases such as hypertrophy and heart failure. After a brief presentation of the cyclic nucleotide pathways in cardiac cells and the major characteristics of the PDE superfamily, this chapter will present their role in cyclic nucleotide compartmentation and the current use of PDE inhibitors in cardiac diseases together with the recent research progresses that could lead to a better exploitation of the therapeutic potential of these enzymes in the future

cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP

Acetylcholine inhibits Ca2+ current by acting exclusively at a site proximal to adenylyl cyclase in frog cardiac myocytes.

1. The effects of acetylcholine (ACh) on the L-type Ca2+ current (ICa) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 microM Iso produced a half-maximal increase in ICa since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 microM ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only approximately 50%. 3. When 10 microM Iso or 30 microM Fsk were applied to a Ca(2+)-free solution on one half of the cell, ICa was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3-10 microM) to the remote part had no effect on ICa, while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates ICa by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of ICa does not involve any additional cAMP-independent mechanisms occurring downstream from cAMP generation

Longitudinal distribution of Na+ and Ca2+ channels and beta-adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes.

1. The distribution of L-type Ca2+ and tetrodotoxin-sensitive Na+ channels and of beta-adrenergic receptors was examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. Rod-shaped cells (250-300 microns long) were sealed at both ends to two patch-clamp pipettes and positioned transversally at different positions between the mouths of two microcapillaries separated by a thin wall. A combination of nifedipine (1 microM) and tetrodotoxin (0.3 microM) (blocking solution) was added to one capillary in order to inhibit macroscopic Ca2+ and Na+ currents (Ica and INa, respectively) in the part of the cell exposed to this capillary. 3. Moving the cell in 10-20 microns steps from the control capillary to the capillary containing the blocking solution induced step decreases in Ica and INa amplitudes. Complete block of both currents occurred when the entire cell was exposed to the blocking solution. 4. Each step decrease in current was due to the loss of activity of the functional Ca2+ and Na+ channels present in the slice of sarcolemmal membrane newly exposed to the blocking solution. These step current changes allowed longitudinal mapping of current density for Ca2+ and Na+ channels on the sarcolemmal membrane. 5. Addition of a submaximal concentration of isoprenaline (10 nM) to the control capillary induced a local increase in Ica which enabled examination of the distribution of functional beta-adrenergic receptors as well. 6. Our results demonstrate that Ca2+ and Na+ channels and beta-adrenergic receptors are equally and essentially uniformly distributed on the sarcolemmal of frog ventricular myocytes

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes.

International audienceThe whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. Thebeta-2 adrenergic agonist zinterol increasedICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 μM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 μM), a nonselective phosphodiesterase inhibitor, or forskolin (10 μM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 μM zinterol. Zinterol had a long lasting action on frogICa because after washout of the drug,ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 μM) during this recovery phase promptly reduced ICaback to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 μM, respectively. In all three preparations, intracellular perfusion with 20 μM PKI(15–22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol onICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase inICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation