1,721,016 research outputs found
The Different Facets of Extracellular Calcium Sensors: Old and New Concepts in Calcium-Sensing Receptor Signalling and Pharmacology
No full textThe current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca2+changes. Widely recognized as a fundamental player in systemic Ca2+homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+sensors is provided
Cardiac cell hypertrophy in vitro: role of calcineurin/NFAT as Ca2+ signal integrators
No full textVarious conditionswere used to investigate the importance of Ca2+ signaling in triggering hypertrophy
in neonatal rat cardiomyocytes in vitro. An increase in cell size and sarcomere reorganization
were induced not only by treatment with receptor agonists, such as angiotensin II, aldosterone,
and norepinephrine, but also by a small depolarization caused by an increase in the KCl concentration
of the medium. This latter treatment has no direct effects on receptor signaling. All these
hypertrophic treatments caused a long-lasting increase in the frequency of spontaneous [Ca2+]
oscillations, causing nuclear translocation of transfected NFAT (GFP). Cyclosporine A inhibited
hypertrophy and NFAT translocation, but not the increased oscillation frequency.We propose here
that calcineurin–NFAT can act as integrators of the Ca2+ signal and can decode alterations in the
frequency even of very rapid Ca2+ oscillations
CADMIUM EFFECTS ON SUBCELLULAR CALCIUM SIGNALLING: A COMPLEX SCENARIO REVEALED BY DIFFERENT FLUORESCENT PROBES
No full textCadmium inhibits acid secretion in stimulated frog gastric mucosa
No full textCadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and
kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate
the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact
amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current
(Isc), transepithelial potential (Vt) and resistance (Rt) were recorded in the continuous presence of cadmium.
Addition of cadmium (20 μM to 1 mM) on the serosal but not luminal side of the mucosae resulted in
inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current.
Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a
cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid
secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in Isc cannot be
explained by an action on: 1) H2 histamine receptor, 2) Ca2+ signalling 3) adenylyl cyclase or 4) carbonic
anhydrase. Conversely, cadmium was ineffective in the presence of the H+/K+-ATPase blocker omeprazole
suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects
the functionality of histamine-stimulated gastric mucosa by inhibiting the H+/K+-ATPase from the
intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant
and may result in new avenues for therapeutic intervention in acute and chronic intoxication
Localization of mitochondrial carnitine/acylcarnitine translocase in sensory neurons from rat dorsal root ganglia
No full textThe carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells
The role of Store-Operated Cyclic AMP Signalling (SOcAMPS) in cardiac physiology and pathology: an in vitro study on neonatal rat cardiomyocytes.
No full textBackground and Aims: Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a recently
identified mechanism of cross-talk between Ca2+ and cAMP signals. In this process, depletion of
Ca2+ in the endoplasmic reticulum (ER) leads to increases in cAMP levels, independently of
cytosolic Ca2+ changes. Expression and functionality of STIM1 (Stromal Interaction Molecule 1), a
transmembrane ER Ca2+ sensor protein, is necessary for SOcAMPS to occur. Interestingly, recent
reports have demonstrated a critical role for STIM1 in the development of cardiac hypertrophy, a
process notoriously controlled both by Ca2+ and cAMP signaling. Here we aimed to evaluate
whether SOcAMPS was manifest in neonatal rat cardiomyocytes and its potential role in cardiac
cell hypertrophy.
Methods: To monitor changes in cAMP levels, real time imaging experiments were performed on
neonatal rat cardiomyocytes transiently transfected with an EPAC-based fluorescent probe for
[cAMP], EPAC H30. Fura-2 and Fluo-4 were used to monitor cytosolic Ca2+ levels and an ER/SR
targeted probe, D1ERcameleon, was used to measure ER [Ca2+]. Long term incubation (48h) of
cardiomyocytes with angiotensin II (1 μM) and aldosterone (1 μM) was used to induce "in vitro"
cell hypertrophy. Increases in cell size and/or sarcomere alignment were monitored microscopically
after labeling with phalloidin-TRITC.
Results: To verify the existence of SOcAMPS in neonatal rat cardiomyocytes, cells were stimulated
in Ca2+-free Ringer's solutions with the low affinity membrane permeant Ca2+ chelator TPEN
(1mM), able to induce a reduction of SR Ca2+ levels ([Ca2+]SR) without affecting cytosolic
[Ca2+]. SR Ca2+ measurements demonstrated that under these experimental conditions, 1 mM
TPEN led to a reduction in intraluminal [Ca2+] that was 50,5±2,4% (8 exp, 11 cells, p<0.001) of
the maximal store depletion. Parallel experiments performed with the EPAC H30 cAMP sensor
showed increases in [cAMP] that were 26,5±3% (13 exp, 13 cells, p<0.001) of the maximum delta
ratio. In the presence of 5 μM Forskolin (FRSK) the TPEN-induced cAMP augmentation resulted
63,7±3,9% of the maximal response (16 exp, 19 cells, p<0.001). Also depletion of SR by the Ca2+
ionophore ionomycin (10 μM) was found to induce significant cAMP increases both in the absence
and presence of FRSK. The participation of STIM1 in the observed phenomenon was proven by the
47 % reduction of the TPEN+FRSK induced [cAMP] signal after transfection of cells with a
shRNA against STIM1 (6 exp, p<0,01). To evaluate the putative role of SOcAMPS in cardiac
hypertrophy, cAMP measurements were performed on angio+aldo treated cells and compared to
control cardiomyocytes. Under these experimental conditions a 20% increase of the TPEN+FRSK
induced response was observed in hypertrophic myocytes (16 exp, p<0,01).
Conclusions: These data straightforwardly establish, for the first time, the existence of SOcAMPS
in the neonatal cardiomyocyte cell model. Also, a significantly increased SOcAMP signalling was
shown to exist in hypertrophic cardiomyocytes. Further experiments to ascertain whether a causeand-
effect relationship exists between SOcAMPS and cardiac cell hypertrophy are in progress
Properties of two forms of lipid depleted bacteriorhodopsin isolated in Phenylsepharos CL-4B chromatography
No full textNew players in pancreatic beta cell signal transduction: which role for the Extracellular Calcium sensing Receptor?
No full textPalmitic acid is associated with halorhodopsin as a free fatty acid. Radiolabeling of halorhodopsin with 3H-palmitic acid and chemical analyses of the reaction products of purofied halorhodopsin with thiols and NaBH4
No full textHalorhodopsin, isolated from Halobacterium, salinarium cells incubated with tritiated palmitic acid, co-elutes with labeled palmitate in phenylsepharose CL-4B chromatography. Halorhodopsin-bound H-3-palmitate is not readily displaced by prolonged exposure to a large excess of detergents and by re-chromatography of radiolabeled halorhodopsin on phenylsepharose. On other hand, the association of labeled palmitate with purified halorhodopsin is not resistant to denaturation induced either by isopropanol/hexane or by SDS gel electrophoresis. We have tested the hypothesis that tightly associated palmitate is bound to halorhodopsin through a thioester bond, which is unstable in denaturing conditions. Using GC/MS, we have analysed the reaction products of native halorhodopsin with specific thioester reagents, thiols and NaBH4, which are inactive on free fatty acids. The results of this analytical approach indicate that there is no thioester bond between halorhodopsin and palmitic acid and that palmitic acid is associated with halorhodopsin as a free fatty acid. (C) 1998 Elsevier Science B.V
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