Calcium-sensitive Fluorescent Probe Research Articles

Degradation Products of Amyloid Protein: Are They The Culprits?

Beta-amyloid (Aβ) peptides are most toxic to cells in oligomeric form. It is commonly accepted that oligomers can form ion channels in cell membranes and allow calcium and other ions to enter cells. The activation of other mechanisms, such as apoptosis or lipid peroxidation, aggravates the toxicity, but it itself can result from the same initial point, that is, ion disturbance due to an increased permeability of membranes. However, experimental studies of membrane channels created by Aβ are surprisingly limited. Here, we report a novel flow cytometry technique which can be used to detect increased permeability of membranes to calcium induced by the exposure to amyloid peptides. Calcium entry into the liposome is monitored using calcium-sensitive fluorescent probe. Undamaged lipid membranes are not permeable to calcium. Liposomes that are prepared in a calcium-free medium become able to accumulate calcium in a calcium-containing medium only after the formation of channels. Using this technique, we demonstrated that the addition of short amyloid fragment Ab25-35, which is known for its extreme toxicity on cultured neurons, readily increased membrane permeability to calcium. However, neither similarly sized peptide Ab22-35 nor full-length peptide Aβ1-42 were producing channels. The formation of channels was observed in the membranes made of phosphatidylserine, a negatively charged lipid, but not in membranes made of the neutral phosphatidylcholine. We have analyzed several issues which could be critical for understanding the pathogenesis of Alzheimer's disease, specifically 1) the need for a negatively charged membrane to produce the ion channel; 2) the potential role of the aggregated form in cellular toxicity of Aβ peptides; 3) channelforming ability of multiple degradation products of amyloid; 4) non-specificity of ion channels formed by amyloid peptides. Potential targets of channel-forming oligomers appear to be intracellular and are organelles well-known for dysfunction in Alzheimer's disease (mitochondria and lysosomes). In fact, lysosomes can also be the producers of degraded amyloid. Provided speculations support the hypothesis that neuronal toxicity can be caused by the degradation products of beta-amyloid.

Blockade of metabotropic glutamate receptor 5 attenuates axonal degeneration in 6-hydroxydopamine-induced model of Parkinson's disease

Although there are numerous strategies to counteract the death of dopaminergic neurons in Parkinson's disease (PD), there are currently no treatments that delay or prevent the disease course, indicating that early protective treatments are needed. Targeting axonal degeneration, a key initiating event in PD, is required to develop novel therapies; however, its underlying molecular mechanisms are not fully understood. Here, we studied axonal degeneration induced by 6-hydroxydopamine (6-OHDA) in vitro and in vivo. We found that metabotropic glutamate receptor 5 (mGluR5) expression increased during 6-OHDA-induced axonal degeneration in primary neurons and that blockade of mGluR5 by its antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 3-[(2-methyl-1, 3-thiazol-4-yl) ethynyl]-pyridine (MTEP) almost completely attenuated the degenerative process in vitro. Furthermore, a rapid increase in intra-axonal calcium levels following 6-OHDA treatment was visualized using a calcium-sensitive fluorescence probe and a calcium chelator prevented the axonal degenerative process induced by 6-OHDA in vitro, whereas application of the mGluR5 antagonist MPEP partially attenuated the increase in intra-axonal calcium. The screening of calcium targets revealed that calpain activation and an increase in phosphorylated extracellular signal-regulated kinase (p-ERK) were calcium dependent during 6-OHDA-induced axonal degeneration in vitro. Consistent with these in vitro findings, blockade of mGluR5 with MPEP attenuated the degeneration of dopaminergic axons induced by 6-OHDA injection into the striatum prior to soma death in the early stage of PD in an in vivo animal model. In addition, MPEP inhibited the increase in mGluR5 expression levels, calpain activation and the elevation of p-ERK in the striatum triggered by 6-OHDA injection in vivo. Taken together, these data identify an mGluR5-calcium-dependent cascade that causes axonal degeneration, and suggest that mGluR5 antagonists could provide effective therapy to prevent the disease process of PD.

SAT-078 Adrenergic Receptor Regulation of Intracellular Calcium in Human Adrenocortical Carcinoma HAC15 Cells

Intracellular calcium fluctuations may play a role in the control of corticosteroid synthesis and secretion by the adrenal cortex. Using live-cell imaging with the calcium-sensitive fluorescent probe Fluo-4 in a human adrenocortical carcinoma cell line (HAC15), we previously demonstrated that activation of M3 muscarinic receptors stimulates intracellular calcium oscillations, enhances aldosterone production, and increases expression of CYP11B2 (1). To further understand autonomic control of cortical cell function, we are investigating the role of adrenergic signalers in regulating calcium homeostasis in HAC15 cells. Activation of adrenergic receptors with the endogenous, non-selective agonists norepinephrine and epinephrine (10μM) causes single, large calcium transients that do not oscillate. A similar calcium response results from the alpha adrenergic agonist phenylephrine, while the beta adrenergic agonist terbutaline does not elicit calcium changes. Consistent with this, norepinephrine-induced calcium changes are blocked by the alpha adrenergic antagonist phentolamine, but not by the beta adrenergic antagonist propranolol. Taken together, these preliminary results suggest that alpha adrenergic receptors are the dominant adrenergic subtype in the human adrenal cortex. Further experiments will use radiolabel binding assays, subtype-selective alpha receptor modulators, and RT-PCR methods, to determine which alpha subtypes are present. Also, ELISA measurements of steroid production will determine the physiological consequence of adrenergic receptor activation.

Discovery of a novel calcium-sensitive fluorescent probe for α-ketoglutarate.

α-Ketoglutarate (α-KG), a pivotal metabolite in energy metabolism, has been implicated in nonalcoholic fatty liver disease (NAFLD) and several cancers. It is recently proposed that plasma α-KG is a surrogate biomarker of NAFLD. Here, we report the development of a novel "turn-on" chemosensor for α-KG that contains a coumarin moiety as a fluorophore. Using benzothiazole-coumarin (BTC) as inspiration, we designed a probe for calcium ion recognition that possesses a unique fluorophore compared with previously reported probes for α-KG measurement. This chemosensor is based on the specific Schiff base reaction and the calcium ion recognition property of the widely used calcium indicator BTC. The probe was synthesized, and a series of parallel experiments were conducted to optimize the chemical recognition process. Compared to the initial weak fluorescence, a remarkable 7.6-fold enhancement in fluorescence intensity (I/I0 at 495 nm) was observed for the conditions in which the probe (1 μmol/L), α-KG (50 μmol/L), and Ca2+ (100 μmol/L) were incubated at 30 °C in EtOH. The probe displayed good selectivity for α-KG even in an environment with an abundance of amino acids and other interfering species such as glutaric acid. We determined that the quantitative detection range of α-KG in EtOH was between 5 and 50 μmol/L. Finally, probe in serum loaded with α-KG (10 mmol/L) showed a 7.4-fold fluorescence enhancement. In summary, a novel probe for detecting the biomarker α-KG through a typical Schiff base reaction has been discovered. With further optimization, this probe may be a good alternative for detecting the physiological metabolite α-KG.

High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.

Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

Characterization of lysophosphatidylcholine-induced changes of intracellular calcium in Drosophila S2 cells

Lysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. LPC could be deacylated to form glycerophosphocholine by neuropathy target esterase (NTE)/Swiss cheese protein (SWS). Although NTE/SWS is important in maintaining Ca(2+) homeostasis, the role of LPC in regulating the intracellular calcium concentration ([Ca(2+)]i) in Drosophila remains poorly understood. We aimed to study the mechanism of LPC-induced [Ca(2+)]i changes in Drosophila S2 cells. The [Ca(2+)]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM. Our results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca(2+)]i in the presence of external calcium ([Ca(2+)]e). The LPC-induced [Ca(2+)]i increase was reduced by 60.7% in the absence of [Ca(2+)]e. Furthermore, the Ca(2+) influx was inhibited by 37.3% after the cells were preincubated with an L-type Ca(2+) channel blocker. In the Ca(2+)-free medium, the LPC-induced [Ca(2+)]i increase was completely blocked using an inositol triphosphate receptor (IP3R) inhibitor. However, a ryanodine receptor (RyR) inhibitor had no effect on the LPC-induced [Ca(2+)]i increase. The LPC-induced [Ca(2+)]i increase in S2 cells was dependent on both the release of Ca(2+) stored in the endoplasmic reticulum and [Ca(2+)]e influx. Both L-type Ca(2+) channels and IP3R might be involved in this process. The LPC-induced [Ca(2+)]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC.

The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase‐D toxin

Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.

A method for detection and characterization of GABA(A) receptor ligands using calcium-sensitive fluorescent probes

A method for detecting and characterizing possible ligands of neuronal GABA(A) receptors has been developed, which is based on measuring the calcium response to GABA by the fluorescence of a two-wavelength Ca-sensitive probe Fura-2. In a young (2–4 days) rat hippocampal cell culture, GABA induced depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to activation of voltage-dependent calcium channels. A brief application of GABA could attenuate the calcium response to a subsequent addition of depolarizing agents (GABA or KCl). However, at modest amplitudes of calcium response to GABA, the reduction of the subsequent effect of KCl was insignificant, and the amplitudes of responses to KCl and to GABA proved to be linearly correlated, with a slope of ∼3.4. Therefore, the GABA calcium signals could be normalized in order to compare experiments performed on different days and different cultures. With such normalization, we estimated the EC50 for GABA in neurons at ∼2.23 μM and the Hill coefficient at ∼1.9. A blocker of voltage-dependent calcium channels nifedipine suppressed the calcium responses both to KCl and to GABA, so that the linear relationship between their amplitudes was retained. To further validate the method, the IC50 and the type of inhibition were verified for known noncompetitive and competitive antagonists of GABA(A) receptors.

Changes In Mitochondrial Permeability Initiate Platelet Phosphatidylserine Exposure In Response to Elevated Cytoplasmic Calcium Levels

AbstractAbstract 2024Platelet phosphatidylserine (PS) exposure and procoagulant activity occur in a subset of activated platelets following stimulation with multiple or high levels of agonists. Key among the upstream regulators of agonist-initiated PS exposure is elevated cytoplasmic calcium levels. Additionally, the importance of mitochondrial permeability transition pore (mPTP) formation in regulating PS exposure has been highlighted by recent studies using cyclophilin D null (CypD -/-) platelets. CypD is a critical regulatory protein in the initiation of mPTP formation, and in the absence of CypD agonist-initiated PS exposure is markedly impaired. An alternative, slower pathway of PS exposure, initiated by the BH3 mimetic ABT-737, is regulated by members of the Bcl-2 protein pathway.It is currently unclear how mitochondrial and calcium-signaling pathways interact to regulate PS exposure in platelets. mPTP formation has been reported as both a consequence and a cause of elevated intracellular calcium levels in other cells. To clarify the relative roles of these two upstream signals, we examined the sequence of events that occur upon platelet activation using calcium and mitochondrial transmembrane potential (ΔΨm) sensitive fluorescent probes. In isolated platelets studied by flow cytometry and among adherent platelets evaluated by confocal microscopy, elevated intracellular calcium levels were observed prior to mPTP formation (as determined by loss of ΔΨm). A rise in mitochondrial calcium levels as measured by rhod-2 occurred coincidentally with elevations in cytoplasmic calcium levels. Similar increases in intracellular calcium levels were observed in CypD -/- and CypD +/+ platelets, despite the almost complete absence of mPTP formation and PS exposure in the CypD -/- platelets. These results are consistent with calcium acting as an upstream signal regulating mPTP formation.Ionomycin (3 μM) stimulation can overcome the defect in PS exposure in CypD -/- platelets. This result has been interpreted to indicate that calcium activates effector pathways downstream of the mPTP. However, an alternative explanation is that ionomycin raises cytoplasmic calcium to an elevated level that is sufficient to initiate mPTP formation even in CypD's absence. Dose response studies indicated that the calcium sensitivity of the mPTP was decreased in CypD -/- platelets, but ionomycin (3uM) caused mPTP opening and PS externalization in both CypD +/+ and CypD -/- platelets. Furthermore, in both CypD +/+ and CypD -/- platelets, mPTP formation and PS exposure were closely correlated at all doses of ionomycin utilized. These results indicate that high doses of ionomycin overcome the CypD -/- defect in PS exposure by initiating non-CypD-dependent mPTP formation.The roles of the mitochondrial and calcium pathways in regulating ABT-737-mediated PS exposure were examined. Extracellular calcium was not required for PS exposure initiated by ABT-737, distinct from its importance in agonist-initiated PS exposure. In contrast to previously reported results (Schoenwaelder et al, Blood 2009) we observed that ABT-737 stimulation causes a loss of ΔΨm coincident with PS exposure. Since disruptions in oxidative metabolism could also cause the observed effect on ΔΨm, ABT-737's effects on mitochondrial permeability were confirmed using a calcein-cobalt quenching assay. Unlike agonist-initiated PS exposure, both increased mitochondrial permeability and PS exposure were not dependent on CypD.Together these results indicate a central role for alterations in mitochondrial membrane permeability in mediating PS exposure. In agonist-stimulated platelets increases in cytoplasmic calcium result in mPTP formation and mitochondrial permeabilization, triggering downstream events that result in PS exposure. In ABT-737 stimulated platelets non-CypD-dependent mitochondrial permeability changes mediated by Bcl-2 proteins may also act as an important initiator of the events that result in PS exposure. Disclosures:No relevant conflicts of interest to declare.

Purification and functional characterization of protoplasts and intact vacuoles from grape cells

BackgroundDuring grape berry ripening, the vacuoles accumulate water, sugars and secondary metabolites, causing great impact in plant productivity and wine quality. However, the molecular basis of these compartmentation processes is still poorly understood. As in many species, the major bottleneck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield. The present paper describes an isolation method of protoplasts and intact vacuoles from grape berry cells and their functional characterization by transport and cytometric assays.FindingsProtoplasts were prepared by enzymatic digestion of grape cells, and vacuoles were released and purified by a Ficoll step gradient centrifugation. The tonoplast stained strongly with the fluorescent dye FM1-43 and most vacuoles maintained an internal acidic pH, as assessed by Neutral Red. Flow cytometry analysis of vacuole samples incubated with the calcium-sensitive fluorescent probe Fluo-4 AM revealed a well-defined sub-population of intact vacuoles. As assessed by the pH-sensitive probe ACMA, intact vacuoles generated and maintained a pH gradient through the activity of V-ATPase and V-PPase and were able to transport Ca2+ via a proton-dependent transport system.ConclusionsHighly pure, intact and functional protoplast and vacuole populations from grape cells were obtained with the present method, which revealed to be fast and efficient. The capacity of the vacuole population to sequester protons and accumulate Ca2+ strongly suggests the intactness and physiological integrity of these extremely fragile organelles. Grapevine protoplasts and vacuoles may be used as models for both basic research and biotechnological approaches, such as proteomics, solute uptake and compartmentation, toxicological assessments and breeding programs.

Differential variations in Ca2+ entry, cytosolic Ca2+ and membrane capacitance upon steady or action potential depolarizing stimulation of bovine chromaffin cells

This study looks into the physiology of the exocytosis of catecholamines released by adrenal medullary chromaffin cells. We have comparatively explored the exocytotic responses elicited by two different patterns of depolarizing stimulation: the widely employed square depolarizing pulses (DPs) and trains of acetylcholine-like action potentials (APs), likely the physiological mode of stimulation in the intact innervated adrenal medulla. APs were applied at 30 Hz, a frequency similar to that produced in a stressful situation. Patch-clamp, cell membrane capacitance, single cell amperometry and fluorescence were the techniques used. The variations of calcium entry measured as the integral of the calcium current, cytosolic calcium (measured with the calcium-sensitive fluorescent probe fluo-4) and exo-endocytosis (membrane capacitance variations) were the parameters measured. Trains of AP depolarizations produced distinct responses compared to those of square depolarizations: (1) Calcium current amplitude decreased to a lesser extent along the AP train; (2) calcium entry and capacitance increments raised linearly with stimulation time whereas they deviated from linearity when square depolarizations were used; (3) slower activation and faster delayed decay phase of cytosolic calcium transients; (4) capacitance increments varied linearly with calcium entry with APs and deviated from linearity with longer depolarizations; (5) little endocytosis after stimulation with longer trains of APs and pronounced endocytosis with longer square depolarizations. Stimulation of chromaffin cells with trains of APs produced patterns of cytosolic calcium transients, exocytotic and endocytotic responses quite different from those elicited by the widely employed DPs. Our study is relevant from the methodological and physiological points of view.

Ionic Calcium Determination in Skim Milk with Molecular Probes and Front-Face Fluorescence Spectroscopy: Simple Linear Regression

The purpose of this study was to determine if the ionic calcium content of skim milk could be determined using molecular probes and front-face fluorescence spectroscopy. Current methods for determining ionic calcium are not sensitive, overestimate ionic calcium, or require complex procedures. Molecular probes designed specifically for measuring ionic calcium could potentially be used to determine the ionic calcium content of skim milk. The goal of the current study was to develop foundation methods for future studies to determine ionic calcium directly in skim milk and other dairy products with molecular probes and fluorescence spectroscopy. In this study, the effect of pH on calcium-sensitive fluorescent probe (Rhod-5N and Fluo-5N) performance using various concentrations of skim milk was determined. The pH of diluted skim milk (1.9 to 8.9% skim milk), was adjusted to either 6.2 or 7.0, after which the samples were analyzed with fluorescent probes (1μM) and front-face fluorescence spectroscopy. The ionic calcium content of each sample was also determined using a calcium ion-selective electrode. The results demonstrated that the ionic calcium content of each sample was highly correlated (R2>0.989) with the fluorescence intensities of the probe-calcium adduct using simple linear regression. Higher than suggested ionic calcium contents of 1,207 and 1,973μM were determined with the probes (Fluo-5N and Rhod-5N) in diluted skim milk with pH 7.0 and 6.2, respectively. The fluorescence intensity of the probe-calcium adduct decreased with a decrease in pH for the same ionic calcium concentration. This study demonstrates that Fluo-5N and Rhod-5N can be used to determine the ionic-calcium content of diluted milk with front-face fluorescence spectroscopy. Furthermore, these probes may also have the potential to determine the ionic calcium content of undiluted skim milk.

Mitochondrial permeability transition in the crustaceanArtemia franciscana: absence of a calcium-regulated pore in the face of profound calcium storage

When mammalian mitochondria are exposed to high calcium and phosphate, a massive swelling, uncoupling of respiration, and release of cytochrome c occur. These changes are mediated by opening of the mitochondrial permeability transition pore (MPTP). Activation of the MPTP in vivo in response to hypoxic and oxidative stress leads to necrotic and apoptotic cell death. Considering that embryos of the brine shrimp Artemia franciscana tolerate anoxia for years, we investigated the MPTP in this crustacean to reveal whether pore opening occurs. Minimum molecular constituents of the regulated MPTP in mammals are believed to be the voltage-dependent anion channel, the adenine nucleotide translocators, and cyclophilin D. Western blot analysis revealed that mitochondria from A. franciscana possess all three required components. When measured with a calcium-sensitive fluorescent probe, rat liver mitochondria are shown to release matrix calcium after addition of >/=100 microM extramitochondrial calcium (MPTP opening), whereas brine shrimp mitochondria continue to take up extramitochondrial calcium and do not release internal stores even up to 1.0 mM exogenously added calcium (no MPTP opening). Furthermore, no swelling of A. franciscana mitochondria in response to added calcium was observed, and no release of cytochrome c could be detected. HgCl(2)-dependent swelling and cytochrome c release were readily confirmed, which is consistent with the presence of an "unregulated pore." Although the absence of a regulated MPTP in A. franciscana mitochondria could contribute to the extreme hypoxia tolerance in this species, we speculate that absence of the regulated MPTP may be a general feature of invertebrates.

Comparison of ionophore induced Ca2+ release in fresh and cryopreserved oocytes

Comparison of ionophore induced Ca2+ release in fresh and cryopreserved oocytes

Calcium signalling in and around the nuclear envelope.

We have compared calcium mobilization by Ins(1,4,5) P(3)(IP(3)), cADP-ribose (cADPR) and nicotinic acid-adenosine dinucleotide phosphate (NAADP) from the envelope of isolated nuclei with the calcium signalling in intact isolated pancreatic acinar cells. Ca(2+) uptake and release were studied with calcium-sensitive fluorescent probes. In the present study, we have shown that all calcium messengers induce Ca(2+) release from the nuclear envelope. Pre-treatment of nuclei with thapsigargin completely abolished the responses to the calcium messengers, indicating that Ca(2+) stores in isolated nuclei are thapsigargin-sensitive. Using different pharmacological tools, we show that Ca(2+) release from pancreatic nuclei is unlikely to occur from stores other than those with endoplasmic reticulum characteristics. We conclude that all three calcium messengers can release Ca(2+) from pancreatic acinar nuclear stores, as previously shown for IP(3) and cADPR. It would appear that NAADP releases Ca(2+) from the same IP(3)- and cADPR-sensitive stores with endoplasmic reticulum characteristics.

The effects of hydroxyethyl starches on intracellular calcium in platelets.

Hydroxyethyl starch (HES) solutions impair platelet function. To determine whether this effect is achieved through interference of HES with intracellular activation processes, in which calcium is the key second messenger, we evaluated the agonist-induced increase of the cytoplasmic calcium concentration in the presence of HES of different molecular weights. Aliquots of citrated whole blood of 12 volunteers were incubated in vitr. with saline, HES 450 (molecular weight in kilodalton), HES 130, and HES 70, resulting in 20% hemodilution. An undiluted sample served as control. The samples were stained with Fluo-3 as the calcium-sensitive fluorescent probe with subsequent flow cytometric analysis. After determination of a baseline, platelets were activated with thrombin receptor activator peptide 6. Platelet activation with thrombin receptor activator peptide 6 resulted in a fast increase in fluorescence (approximately eightfold), representing intracellular calcium mobilization. None of the tested HES solutions exerted a statistically significant effect on the cytoplasmic calcium concentration compared with samples that were incubated with saline or that remained undiluted. These results indicate that the known inhibiting effect of HES on platelets is not achieved through interference with intracellular activation processes. Hydroxyethyl starch does not exert its known inhibitory effect on platelet function by interfering with intracellular activation processes.

Muscarinic signalling affects intracellular calcium concentration during the first cell cycle of sea urchin embryos

The existence of a response to acetylcholine (ACh) and cholinomimetic drugs in sea urchin eggs and zygotes was investigated in two sea urchin species: Paracentrotus lividus and Lytechinus pictus. The calcium sensitive fluorescent probe, Fura-2 dextran, was employed to investigate the regulation of cytosolic free calcium concentration ([Ca 2+] i) by cholinomimetic drugs in unfertilised and fertilised eggs of both the sea urchin species. Exposure to cholinomimetic agonists/antagonists, either extracellularly or intracellularly, had no effect either on resting [Ca 2+] i levels in the unfertilised sea urchin egg, or on the transient [Ca 2+] i increase at fertilisation. However, following fertilisation, extracellular application of ACh receptors agonists, such as ACh and carbachol, predominantly muscarinic agonist, but not nicotine, induced a significant increase in [Ca 2+] i, which was partially inhibited by atropine. As a consequence of exposure after fertilisation to the agonists of ACh receptors, chromatin structure was transiently affected. The hypothesis is proposed that muscarinic receptors may be involved in the (presumably Ca 2+-dependent) modulation of the nuclear status during the first cell cycles.

Adenosine 5′-triphosphate-evoked calcium responses in terminal Schwann cells of lanceolate sensory endings isolated from rat vibrissae

Extracellular adenosine 5′-triphosphate (ATP) has been known to mediate and modulate cutaneous sensations. We examined the effect of this substance on isolated terminal Schwann cells associating with lanceolate endings, the mechanoreceptors of rat vibrissae. The free intracellular calcium concentration ([Ca 2+] i) of the sensory device was monitored by digital image microscopy in combination with a calcium-sensitive fluorescent probe, Fura-2. Application of ATP in concentrations raging from 10 μM to 1 mM evoked an increase in [Ca 2+] i in Schwann cell processes covering the lancet-like axon terminals as well as in round perikarya of the cells protruding from the terminals. In both portions, the ATP-evoked [Ca 2+] i elevations were slowly oscillatory at 10 and 20 μM, and continuous at concentrations higher than 50 μM. Suramin 100 μM blocked the effect of ATP. Uridine 5′-triphosphate was equipotent with ATP, while α,β-methylene ATP was ineffective. These data indicate that the terminal Schwann cells express P2Y purinoceptors linked with the intracellular Ca 2+ signaling, and that this phenomenon is involved in the ATP-mediated sensory modulation.

Ryanodine receptor-mediated [Ca 2+] i release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

The hypothesis that intracellular calcium ([Ca 2+] i) release in glomus cells via ryanodine receptor (RyR) activation by caffeine may be independent of natural stimuli and chemosensory discharge was tested in the rat carotid body (CB). CB type I cells were isolated, plated and preloaded with calcium-sensitive fluorescent probe, Indo-1AM. With the increase of caffeine dose (0–50 mM) cytosolic calcium ([Ca 2+] c) increased from 85±15 nM to 1933±190 nM ( n=6) at normoxia (P o 2=125–130 Torr, P co 2=25–30 Torr, pH 7.30–7.35). Hypoxia (P o 2=10–15 Torr) increased and hypocapnia (P co 2=7–9 Torr) decreased the cytoplasmic calcium [Ca 2+] c levels, independent of caffeine. Caffeine-related [Ca 2+] c increase was the same in the presence and the absence of extracellular calcium ([Ca 2+] o), indicating the source of Ca 2+ ions is the cellular store. Permeabilization of the cell membrane with saponin (25 μg/ml) retained the caffeine response. Additional treatment of the cells with 50 μM ryanodine (an inhibitor of the caffeine-activated RyR site) abolished caffeine-stimulated response. In vitro CB chemosensory (carotid sinus nerve, CSN) responses to hypoxia (P o 2=35–40 Torr) were not altered by caffeine. These results suggest that [Ca 2+] i stores in CB cells, mobilized by RyR activation, do not participate in the CSN responses to natural stimuli.

The influence of beta-adrenergic activation on noradrenergic alpha1 activation of rabbit afferent arterioles.

The aim of the present investigation was to examine the effect of beta-adrenergic stimulation on smooth muscle calcium concentration ([Ca2+]i) in resting conditions and after administration of norepinephrine (NE) to stimulate alpha-adrenoceptors in isolated rabbit afferent arterioles loaded with the calcium-sensitive fluorescent probe fura-2. [Ca2+]i was evaluated in the proximal and distal parts of the arterioles. NE (4x10(-7) M) increased [Ca2+]i in both these regions. The alpha1-adrenoceptor antagonist prazosin (10(-7) M) totally abolished the NE-induced increase in [Ca2+]i, while the alpha2-adrenoceptor antagonist yohimbine (5x10(-7) M) had no influence on the response to NE. When beta-adrenoceptors were stimulated, using isoproterenol (10(-7) M), the NE-induced increase in [Ca2+]i was significantly lower in both regions. Activation of beta-adrenoceptors with isoproterenol did not affect the [Ca2+]i increase in response to depolarization with K+. Since beta-adrenoceptor stimulation raises the smooth muscle cell levels of cAMP, an adenylate cyclase stimulator, forskolin (10(-5) M) was administered prior to NE application. This maneuver also blunted the increase in [Ca2+]i in both regions. We conclude that the calcium response to NE in the isolated rabbit afferent arteriole is mediated by an alpha1-adrenoceptor. beta-Adrenoceptor stimulation and forskolin blunt the increase in [Ca2+]i induced by NE stimulation, indicating that cAMP counteracts the NE-induced activation of alpha1-adrenoceptors.