thapsigargin and fura-2-am

In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy.

Topics: Animals; Azoles; Cadmium; Calcium; Catecholamines; Cattle; Cells, Cultured; Chromaffin Cells; Cytosol; Enzyme Inhibitors; Exocytosis; Fura-2; Glycolipids; Membrane Transport Modulators; Nifedipine; Nitrobenzenes; Potassium; Sodium; Sodium Channels; Tetrodotoxin; Thapsigargin

Topics: Calcium; Calcium Signaling; Cell Line; Cell Membrane; Fluorescent Dyes; Fura-2; Humans; Kidney; Membrane Proteins; Microscopy, Fluorescence; Neoplasm Proteins; Stromal Interaction Molecule 1; Thapsigargin; Transfection

The phytocannabinoid cannabidiol (CBD) is at the forefront of therapeutic cannabinoid research due to its non-psychotropic properties. Research supports its use in a variety of disorders, yet the cellular mechanisms of its action remain unclear. In this study, the effect of CBD upon Ca2+ homeostasis in hippocampal cells was characterised. CBD (1 microM) elevated intracellular Ca2+ ([Ca2+]i) by approximately +45% of basal Ca2+ levels in both glia (77% responders) and neurones (51% responders). Responses to CBD were reduced in high excitability HEPES buffered solution (HBS), but not affected in low excitability/low Ca2+ HBS. CBD responses were also significantly reduced (by 50%) by the universal Ca2+ channel blocker cadmium (50 microM) and the L-type specific Ca2+ channel blocker nifedipine (20 microM). Interestingly, intracellular store depletion with thapsigargin (2 microM) had the most dramatic effect on CBD responses, leading on average to a full block of the response. Elevated CBD-induced [Ca2+]i responses (>+100%) were observed in the presence of the CB1 receptor antagonist, AM281 (1 microM), and the vanilloid receptor antagonist, capsazepine (CPZ, 1 microM). Overall, our data suggest that CBD modulates hippocampal [Ca2+]i homeostasis via intracellular Ca2+ stores and L-type VGCC-mediated Ca2+ entry, with tonic cannabinoid and vanilloid receptor signalling being negatively coupled to this pathway.

Topics: Animals; Animals, Newborn; Cadmium; Calcium; Calcium Channel Blockers; Cannabidiol; Cells, Cultured; Drug Interactions; Enzyme Inhibitors; Fura-2; Hippocampus; Intracellular Space; Models, Neurological; Morpholines; Neuroglia; Neurons; Nifedipine; Pyrazoles; Rats; Rats, Sprague-Dawley; Thapsigargin; Time Factors

The effect of the environmental toxicant nonylphenol on cytosolic free Ca2+ concentration ([Ca2+]i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether nonylphenol alters Ca2+ levels and causes cell death in MG63 human osteosarcoma cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Nonylphenol at concentrations above 3 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+. The nonylphenol-induced Ca2+ influx was insensitive to blockade of L-type Ca2+ channel blockers. After pretreatment with 10 microM nonylphenol, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to induce [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change nonylphenol-induced [Ca2+]i rises. The nonylphenol-induced [Ca2+]i rises were enhanced or inhibited by phorbol myristate acetate or GF 109203X, respectively. At concentrations of 10 and 20 microM nonylphenol killed 55% and 100% cells, respectively. The cytotoxic effect of 10 microM nonylphenol was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, nonylphenol induced [Ca2+]i rises by causing Ca2+ release from intracellular stores and Ca2+ influx from extracellular space. Furthermore, nonylphenol can cause Ca2+-unrelated cytotoxicity in a concentration-dependent manner.

Topics: Calcium; Calcium Channel Blockers; Cell Death; Cell Line, Tumor; Cell Proliferation; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Estrenes; Fluorescent Dyes; Fura-2; Humans; Osteoblasts; Osteosarcoma; Phenols; Pyrrolidinones; Spectrometry, Fluorescence; Tetrazolium Salts; Thapsigargin; Type C Phospholipases

Galantamine is currently used to treat Alzheimer's disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer's disease.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Apoptosis; Blotting, Western; Bungarotoxins; Calcium; Cattle; Cell Line, Tumor; Cholinesterase Inhibitors; Chromaffin Cells; Drug Interactions; Enzyme Inhibitors; Flow Cytometry; Fura-2; Galantamine; Humans; Immunohistochemistry; Neuroblastoma; Nicotine; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, Nicotinic; Thapsigargin

We investigated the effects of ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], a myosin light chain kinase (MLCK) inhibitor, on intracellular Ca2+ concentration ([Ca2+]i), contraction induced by high K+ and an agonist, and capacitative Ca2+ entry in fura-2-loaded guinea pig tracheal smooth muscle. ML-9 inhibited both the increase in [Ca2+]i and the contraction induced by 60 mM K+, 1 microM methacholine or 1 microM thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. However, another MLCK inhibitor, wortmannin (3 microM), inhibited the contraction elicited by these stimuli without affecting [Ca2+]i. Under the condition that the thapsigargin-induced contraction was fully suppressed by 3 microM wortmannin, 30 microM ML-9 caused a further decrease in [Ca2+]i. The inhibitory effects of ML-9 on [Ca2+]i and the contraction elicited by methacholine were similar to those of SKF-96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), a Ca2+ channel blocker. These results indicate that ML-9 acts as a potent inhibitor of Ca2+-permeable channels independently of MLCK inhibition in tracheal smooth muscle.

Topics: Androstadienes; Animals; Azepines; Calcium; Calcium Channel Blockers; Fura-2; Guinea Pigs; Imidazoles; In Vitro Techniques; Isometric Contraction; Male; Methacholine Chloride; Muscarinic Agonists; Muscle, Smooth; Myosin-Light-Chain Kinase; Potassium; Potassium Channel Blockers; Thapsigargin; Trachea; Wortmannin

The olfactory epithelium (OE) is composed of olfactory sensory neurons (OSNs) and sustentacular cells; it lies in the nasal cavity where it is protected by a thin mucus layer. The finely regulated composition of this mucus provides OSN with a suitable ionic environment. To maintain the functional integrity of the epithelium despite permanent physical, chemical and microbial aggressions, both OSNs and surrounding sustentacular cells are continuously renewed from globose basal cells. Moreover, the sense of smell is involved in so numerous behaviours (feeding, reproduction, etc.) that it has to cross-talk with the endocrine and neuroendocrine systems. Thus, besides its sensory function, the olfactory epithelium is thought to undergo a lot of complex regulatory processes. We therefore studied the effects of various neuropeptides on primary cultures of Sprague-Dawley rat olfactory epithelium cells. We found that arginine-vasopressin (AVP) triggered a robust, dose-dependent calcium increase in these cells. The cell response was essentially ascribed to the V1a AVP receptor, whose presence was confirmed by RT-PCR and immunolabelling. In the culture, V1a but not V1b receptors were present, mainly localized in neurons. In the epithelium, both subtypes were found differentially distributed. V1a-R were localized mainly in globose basal cells and at the apical side of the epithelium, in the area of the dendritic knobs of OSNs. V1b-R were strongly associated with Bowman's gland cells and globose basal cells. These localizations suggested potential multifaceted roles of a hormone, AVP, in the olfactory epithelium.

Topics: Animals; Animals, Newborn; Arginine Vasopressin; Boron Compounds; Cadmium Chloride; Calcium; Calcium Channel Blockers; Cells, Cultured; Chelating Agents; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; Estrenes; Extracellular Space; Fura-2; Immunohistochemistry; Male; Neurons; Olfactory Mucosa; Oxytocin; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Oxytocin; Receptors, Vasopressin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thapsigargin; Time Factors; Verapamil

Pathogenic Naegleria fowleri amebae are resistant to the lytic effects of serum complement. The presence of surface glycoproteins or removal of the membrane attack complex (MAC) of complement from the cell surface by vesiculation serve to protect the amebae from complement lysis. The specific mediators important in stimulating complement resistance are not defined. These studies were undertaken to examine the effect of Ca(2+) ions in initiating complement resistance of N. fowleri in contrast to non-pathogenic complement-sensitive N. gruberi. Chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) or chelation of intracellular calcium with 1,2-bis-(O-Aminophenoxy) ethane-N,N,N,N tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) increased complement lysis of N. fowleri. Chelation of calcium ions did not affect complement sensitivity of N. gruberi. Increased lysis of ionomycin-treated N. fowleri was detected after exposure to serum complement, suggesting that a threshold level of Ca(2+) mediates complement resistance before survival mechanisms are overwhelmed and lysis occurs. A differential influx of Ca(2+) ions occurred in fura-2 labeled N. fowleri after deposition of complement component C9 to form the MAC complex on the cell surface in comparison to N. gruberi. These studies suggest that Ca(2+) ions influence complement resistance in N. fowleri but do not play a role in altering the sensitivity of N. gruberi to complement.

Topics: Animals; Calcium; Chelating Agents; Complement Activation; Complement System Proteins; Egtazic Acid; Fluorescent Dyes; Fura-2; Humans; Ion Transport; Ionomycin; Ionophores; Magnesium; Manganese; Naegleria fowleri; Signal Transduction; Thapsigargin

Reported species differences in the stimulus-secretion coupling of insulin release made it important to compare the Ca2+ handling of rat beta-cells with that previously observed in mice. Single beta-cells and small aggregates were prepared from pancreatic islets of Wistar rats, attached to cover slips and then used for measuring the cytoplasmic Ca2+ concentration ([Ca2+]i) with the ratiometric fura-2 technique. Glucose (11 mM) induced slow oscillations of [Ca2+]i similar to those seen in other species, including humans. Comparison of the oscillations in rat beta-cells with those previously described in mouse revealed that there was a slightly lower frequency and an increased tendency to transformation into sustained [Ca2+]i in response to glucagon or caffeine. Ryanodine (5-20 microM) did not affect existing oscillations but sometimes restored rhythmic activity in the presence of caffeine. Stimulation with glucose resulted not only in oscillations but also in transients of [Ca2+]i sometimes appearing in synchrony in adjacent beta-cells and disappearing after the addition of 200 nM thapsigargin or 20 mM caffeine. The frequency of transients recorded in a medium containing glucagon and methoxyverapamil was higher than seen under similar conditions in mouse beta-cells. Although exhibiting some differences compared with mouse beta-cells, rat beta-cells also have an intrinsic ability to oscillate and to generate the transients of [Ca2+] that are supposed to synchronize the rhythmicity of the islets in the pancreas.

Topics: Animals; Caffeine; Calcium; Fura-2; Glucagon; Glucose; Islets of Langerhans; Male; Phosphodiesterase Inhibitors; Rats; Rats, Wistar; Ryanodine; Thapsigargin

The effect of estrogens (diethylstilbestrol [DES], 17 beta-estradiol) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in hormone-insensitive PC3 human prostate cancer cells was examined.. [Ca(2+)](i) changes in suspended cells were measured by using the Ca(2+)-sensitive fluorescent dye fura-2.. Estrogens (1--20 microM) increased [Ca(2+)](i) concentration-dependently with DES being more potent. Ca(2+) removal inhibited 50 +/- 10% of the signal. In Ca(2+)-free medium, pretreatment with 20 microM estrogens abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), but pretreatment with CCCP and thapsigargin did not alter DES-induced Ca(2+) release and partly inhibited 17 beta-estradiol-induced Ca(2+) release. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 1- 20 microM estrogens in Ca(2+)-free medium. Pretreatment with 1 microM U73122 to block phospholipase C-coupled inositol 1,4,5-trisphosphate formation did not alter estrogens-induced Ca(2+) release. The effect of 20 microM estrogen on [Ca(2+)](i) was not affected by pretreatment with 0.1 microM estrogens.. Estrogen induced significant Ca(2+) release and Ca(2+) influx in an inositol 1,4,5-trisphosphate-independent manner in PC3 cells. These effects of estrogens on Ca(2+) signaling appear to be nongenomic. Prostate 47:141-148, 2001.

Topics: Antineoplastic Agents, Hormonal; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Diethylstilbestrol; Enzyme Inhibitors; Estradiol; Estrenes; Fluorescent Dyes; Fura-2; Humans; Male; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Pyrrolidinones; Thapsigargin; Tumor Cells, Cultured; Uncoupling Agents

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.

Topics: Adenosine Diphosphate Ribose; ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Antigens, CD; Antigens, Differentiation; Apoptosis; Calcium; Calcium Signaling; Cell Count; Cell Division; Cell Line; Chelating Agents; Cyclic ADP-Ribose; Enzyme Activation; Enzyme Inhibitors; Extracellular Space; Fluorescent Dyes; Fura-2; Humans; Membrane Glycoproteins; Microscopy, Video; NAD; NAD+ Nucleosidase; Osteoblasts; Ryanodine; Stimulation, Chemical; Thapsigargin

Ligation of macrophage alpha(2)-macroglobulin signalling receptors (alpha(2)MSR) with activated alpha(2)-macroglobulin (alpha(2)M*) increases intracellular Ca(2+), and cytosolic phospholipase A(2) (cPLA(2)) and phospholipase D activities. In view of the relationship between cellular Ca(2+) and mitogenesis, we examined the effect of the product of cPLA(2) activity, arachidonic acid (AA), on nuclear Ca(2+) levels in macrophages stimulated with alpha(2)M*, platelet derived growth factor, and bradykinin. AA addition increased Ca(2+) levels in Fura-2/AM loaded nuclei from both buffer-treated and agonist-stimulated cells, but the increase in stimulated macrophages was 2-4-fold higher. Preincubation of Fura-2/AM loaded nuclei with EGTA or BAPTA/AM abolished AA-induced increase in nuclear Ca(2+) levels. Preincubation of nuclei with indomethacin did not affect AA-induced increase in nuclear Ca(2+) in agonist-stimulated nuclei. It is concluded that in macrophages stimulated with various agonists, AA, derived from cPLA(2)-dependent hydrolysis of phospholipids, plays a significant role in regulating nuclear Ca(2+) levels and thus nuclear functions.

Topics: alpha-Macroglobulins; Arachidonic Acid; Bradykinin; Calcium; Cell Nucleus; Chelating Agents; Cyclooxygenase Inhibitors; Cytoplasm; Egtazic Acid; Enzyme Inhibitors; Extracellular Space; Fluorescent Dyes; Fura-2; Indomethacin; Low Density Lipoprotein Receptor-Related Protein-1; Macrophages; Phospholipase D; Phospholipases A; Platelet-Derived Growth Factor; Receptors, Immunologic; Signal Transduction; Thapsigargin

Cotinine is the major metabolite of nicotine. It has some biological activity, but its pathophysiological effects are largely unclear. We studied whether cotinine initiates calcium transients or affects those induced by nicotine. In bovine adrenal chromaffin cells labeled with the fluorescent calcium indicator Fura 2, cotinine (0. 32-3.2 mM) concentration-dependently increased the intracellular Ca(2+) concentration ([Ca(2+)](i)). The effect was abolished by omitting extracellular Ca(2+) during the stimulations. Also nicotinic receptor channel blockers hexamethonium (10 microM-1 mM) and chlorisondamine (100 microM), as well as a competitive nicotinic receptor antagonist dihydro-beta-erythroidine (10-100 microM), inhibited the response. Cotinine (0.32-3.2 mM) preincubation for 2 min inhibited both the nicotine-induced and the cotinine-induced increases in [Ca(2+)](i). Also nicotine (3.2-10 microM) inhibited the cotinine-induced increase in [Ca(2+)](i). Tetrodotoxin (1 microM) and thapsigargin (1 microM) pretreatments did not affect the responses to cotinine, while 300 nM nimodipine partially inhibited the cotinine-induced increase in [Ca(2+)](i). The results indicate that cotinine has nicotine-like effects on chromaffin cells. It may also desensitize the nicotinic cholinergic receptors, possibly by acting as a low-affinity agonist at these receptors.

Topics: Animals; Calcium; Cattle; Chromaffin Cells; Cotinine; Fluorescent Dyes; Fura-2; Hexamethonium; Nicotine; Nicotinic Agonists; Nimodipine; Tetrodotoxin; Thapsigargin

Homocysteine found in the plasma of patients with coronary heart disease, induces vascular smooth muscle cell (VSMC) proliferation and increases deposition of extracellular matrix (ECM) components. Yet, the mechanism by which homocysteine mediates this effect and its role in vascular disease is largely unknown. We hypothesized that homocysteine induces ECM production via intracellular calcium release in VSMC. To test this hypothesis, aortic VSMC from Sprague-Dawley rats were isolated and characterized by positive labeling for vascular smooth muscle alpha-actin. Early passage cells (p2-3) were grown in monolayer on coverslips. Calcium transients were quantified with fura2/AM spectrofluorometry. Homocysteine induced intracellular calcium [Ca(2+)](i) transients with an EC(50) of 60 +/- 5 nM. The EC(50) for glutathione and cysteine were 10 and 100-fold lower, respectively. Depleting extracellular calcium did not alter the homocysteine effect on intracellular calcium; however, thapsigargin pretreatment, which depletes intracellular Ca(2+) stores, abolished the homocysteine effect, demonstrating its dependence on intracellular Ca(2+) stores. Extracellular sodium depletion significantly (P < 0.05) increased [Ca(2+)](i) also suggesting a possible role of sodium-calcium exchange in the process. To begin to elucidate the intracellular pathways by which homocysteine might act, VSMC were pretreated with specific inhibitors and stimulators prior to homocysteine stimulation. Staurosporine and phorbol myrisate acetate (PMA), potent simulators of protein kinase C, augmented the release of Ca(2+) by homocysteine. Interestingly, pretreatment with the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) greatly exacerbated the sensitivity of VSMC to homocysteine. In contrast, pretreatment with either the phospholipase A(2) activator neomycin, the antioxidant and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor, pravastatin, the tyrosine kinase inhibitor genestein, or the calcium channel blocker, felodipine completely inhibited the homocysteine-induced Ca(2+) signal in VSMC. This suggests the role of multiple signaling pathways in the homocysteine effect on VSMC Ca(2+). Effects of homocysteine on collagen production, as ascertained by immunoblot analysis, correlated with its effect in intracellular calcium. Regardless of the signaling pathways involved, homocysteine, by virtue of its role on VSMC proliferation and ECM deposition, ha

Topics: Angiotensin II; Animals; Aorta; Calcium; Cells, Cultured; Collagen; Felodipine; Fluorescent Dyes; Fura-2; Glutathione; Homocysteine; Homocystine; Kinetics; Muscle, Smooth, Vascular; Neomycin; NG-Nitroarginine Methyl Ester; Pravastatin; Rats; Second Messenger Systems; Staurosporine; Tetradecanoylphorbol Acetate; Thapsigargin

Agonist-stimulated Ca2+ influx is critically important to mediate the function of endothelial cells. It has been suggested that release of Ca2+ from internal stores activates Ca2+ influx across the plasma membrane. In the present study, we investigated the effects of ML-9, a myosin light-chain kinase (MLCK) inhibitor, and genistein, a tyrosine kinase inhibitor, on the agonist stimulated Ca2+ response in porcine aortic endothelial cells loaded with a Ca2+-sensitive dye, fura-2. ML-9 almost completely abolished Ca2+ influx, whereas genistein only partially attenuated Ca2+ entry. Both of them did not affect the mobilization of Ca2+ from internal stores. In contrast, genistein was more potent in the inhibition of Mn2+ influx than ML-9. These findings indicate the different selectivity for Ca2+ and Mn2+ in the cation entry pathway in agonist-stimulated endothelial cells.

Topics: Animals; Aorta; Azepines; Bradykinin; Calcium; Cations, Divalent; Cell Membrane; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Genistein; Isoflavones; Kinetics; Manganese; Myosin-Light-Chain Kinase; Protein-Tyrosine Kinases; Swine; Thapsigargin

1. A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin-evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]i), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin-evoked divalent cation-permeable pathway was activated during, but not prior to, release from internal stores. 3. Thapsigargin (1 microM) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca(2+)-influx mechanism. The amplitude of the thrombin- and thapsigargin-induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient. 4. In whole-cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range -110 to +60 mV and was blocked by 1 mM Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+ > > Ba2+ > Mn2+, Mg2+. The thapsigargin-evoked current density at -100 mV varied between 0.42 and 2.1 pA pF-1 in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin-induced inward current had fully developed. 5. These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca(2+)-store-release-activated Ca2+ current (Icrac) rather than via receptor-operated or second messenger-dependent Ca2+ channels. The level of expression of Icrac appears to be a major factor in determining the duration of the thrombin-evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i-dependent events.

Topics: Calcium; Cations, Divalent; Electrophysiology; Fluorescence; Fluorescent Dyes; Fura-2; Humans; Ion Transport; Leukemia, Erythroblastic, Acute; Manganese; Patch-Clamp Techniques; Signal Transduction; Thapsigargin; Thrombin; Tumor Cells, Cultured

The effects of the WT on store-mediated Ca2+ entry and protein tyrosine phosphorylation were investigated in fura-2-loaded human platelets. Wortmannin (2 microM) attenuated the rise in [Ca2+]i caused by Ca2+ entry while having no effect on the mobilisation of Ca2+ from internal stores. It also reduced store-depletion-evoked protein tyrosine phosphorylation. These findings demonstrate that WT is an inhibitor of tyrosine phosphorylation and store-mediated calcium entry and provide further evidence for the involvement of a tyrosine phosphorylation step in the link between Ca2+ store depletion and Ca2+ influx in human platelets.

Topics: Androstadienes; Blood Platelets; Blood Proteins; Calcium; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; In Vitro Techniques; Kinetics; Phosphodiesterase Inhibitors; Phosphoproteins; Phosphorylation; Phosphotyrosine; Terpenes; Thapsigargin; Wortmannin

Fatty acids with more than 22 carbon atoms (very-long-chain fatty acids; VLCFAs) are normal cellular components that have been implicated in the pathophysiology of a number of peroxisomal disorders. To date, however, essentially nothing is known regarding their biological activities. Ca2+ mobilization is an important intracellular signalling system for a variety of agonists and cell types. Given that several polyunsaturated long-chain fatty acids mobilize intracellular Ca2+ and that we have postulated that the VLCFAs may be involved in signal transduction, we examined whether the tetraenoic VLCFA induced Ca2+ mobilization in human neutrophils. We report that fatty acid-induced intracellular Ca2+ mobilization declined for fatty acid species of more than 20 carbon atoms, but increased again as the carbon chain length approached 30. This Ca2+ mobilization occurred independently of inositol 1,4,5-triphosphate production and protein kinase C translocation and involved both the release of Ca2+ from the intracellular stores and changes to the influx or efflux of the ion. We further observed that triacontatetraenoic acid [30:4(n-6)] mobilized Ca2+ from a thapsigargin-insensitive intracellular pool distinct from the thapsigargin-sensitive pools affected by arachidonic acid [20:4(n - 6)] or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). 20:4 (n - 6) induced strong superoxide production (chemiluminescence) which was inhibited by thapsigargin pretreatment. In contrast, fatty acid-induced superoxide production progressively declined as the carbon chain length increased beyond 20-22 carbon atoms. Further studies suggested that the thapsigargin-insensitive Ca2+ mobilization elicited by 30:4 (n - 6) was not related to oxyradical formation, while the thapsigargin-sensitive Ca2+ mobilization induced by 20:4 (n - 6) may be involved in the initiation but not necessarily the maintenance of superoxide production. In conclusion, this is the first report to demonstrate a biological activity for the VLCFA and indicates that 30:4 (n - 6) influences second messenger systems in intact cells that differ from those affected by long-chain fatty acids such as 20:4 (n - 6).

Topics: Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Fatty Acids; Fura-2; Humans; Inositol 1,4,5-Trisphosphate; Luminescent Measurements; Neutrophils; Ovalbumin; Protein Kinase C; Second Messenger Systems; Serum Albumin, Bovine; Structure-Activity Relationship; Superoxides; Terpenes; Thapsigargin

1. The effects of hyperosmotic stress on cytosolic calcium concentration ([Ca2+]i) were studied by ratio image analysis in single cells of an osteoblast-like bone cell line (RCJ 1.20) loaded with fura-2 AM. 2. The ratio (340 nm/380 nm) of steady-state [Ca2+]i in resting osteoblasts kept in Hepes-buffered medium was 0.82 +/- 0.04. A hyperosmotic stimulus (200 mosmol l-1 sucrose) produced a [Ca2+]i transient with a peak ratio of 1.28 +/- 0.09, which decayed with an apparent half-life (t1/2) of 42.7 +/- 2.6 s. 3. The hyperosmotically induced [Ca2+]i transients were insensitive to verapamil, diltiazem or nifedipine, which excludes the involvement of dihydropyridine-sensitive Ca2+ channels in the process. Non-specific Ca2+ channel blockers (Mn2+, Ni2+, La3+ or Gd3+) partially abolished the hyperosmotically induced [Ca2+]i elevation, indicating the contribution of extracellular Ca2+ influx. 4. A hyperosmotic stimulus applied in Ca(2+)-free medium (0.5 mM EGTA) lowered the [Ca2+]i peak to a ratio of 0.96 +/- 0.08 (P < 0.001) compared with a Ca(2+)-containing medium. This suggests that the [Ca2+]i increase is due to extracellular influx, as well as release from an intracellular Ca2+ pool. 5. Application of thapsigargin (0.5 microM), a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, in Ca(2+)-free medium caused transient [Ca2+]i elevation to peak ratios of 1.33 +/- 0.09, and completely abolished the [Ca2+]i response to a hyperosmotic stimulus. This implies the existence of a thapsigargin-sensitive intracellular pool of Ca2+ that is mobilized by hyperosmotic stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cell Line; Cytoskeleton; Cytosol; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Osmolar Concentration; Osteoblasts; Rats; Terpenes; Thapsigargin

Cross-linking the high affinity IgE receptor, Fc epsilon R1, with multivalent antigen induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent release of intracellular Ca2+ stores, Ca2+ influx, and secretion of inflammatory mediators from RBL-2H3 mast cells. Here, fluorescence ratio imaging microscopy was used to characterize the antigen-induced Ca2+ responses of single fura-2-loaded RBL-2H3 cells in the presence and absence of extracellular Ca2+ (Ca2+o). As antigen concentration increases toward the optimum for secretion, more cells show a Ca2+ spike or an abrupt increase in [Ca2+]i and the lag time to onset of the response decreases both in the presence and the absence of Ca2+o. When Ca2+o is absent, fewer cells respond to low antigen and the lag times to response are longer than those measured in the presence of Ca2+o, indicating that Ca2+o contributes to Ca2+ stores release. Ins(1,4,5)P3 production is not impaired by the removal of Ca2+o, suggesting that extracellular Ca2+ influences Ca2+ stores release via an effect on the Ins(1,4,5)P3 receptor. Stimulation with low concentrations of antigen can lead, only in the presence of Ca2+o, to a small, gradual increase in [Ca2+]i before the abrupt spike response that indicates store release. We propose that this small, initial [Ca2+]i increase is due to receptor-activated Ca2+ influx that precedes and may facilitate Ca2+ stores release. A mechanism for capacitative Ca2+ entry also exists in RBL-2H3 cells. Our data suggest that a previously undescribed response to Fc epsilon R1 cross-linking, inhibition of Ca2+ stores refilling, may be involved in activating capacitative Ca2+ entry in antigen-stimulated RBL-2H3 cells, thus providing the elevated [Ca2+]i required for optimal secretion. The existence of both capacitative entry and Ca2+ influx that can precede Ca2+ release from intracellular stores suggests that at least two mechanisms of stimulated Ca2+ influx are present in RBL-2H3 cells.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cations, Divalent; Cell Line; Dinitrophenols; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Ion Transport; Mast Cells; Rats; Receptors, IgE; Serum Albumin, Bovine; Terpenes; Thapsigargin

We investigated the effect of cathepsin G, a serine protease in polymorphonuclear granulocytes, on the function of human lymphocytes. Cathepsin G increased the [3H]thymidine incorporation into human lymphocytes. This mitogenic activity was due to the proteolytic activity of cathepsin G. Both B and T cells showed increased [3H]thymidine incorporation, and this effect was more remarkable for T cells than for B cells. Among the T cell subsets, CD4+ T cells showed the increase in DNA synthesis, but CD8+ T cells did not. When human lymphocytes were stimulated with cathepsin G, intracellular free Ca2+ concentration ([Ca2+]i) increased in B and T cells, including CD4+ T cells and CD8+ T cells. The change in intracellular Ca2+ was due to Ca2+ influx and release of intracellular stores. Cathepsin G also induced the production of inositol 1,4,5-trisphosphate (IP3) in B cells, CD4+ T cells, and CD8+ T cells, leading to the release of Ca2+ from intracellular stores. Moreover, the stimulation with cathepsin G resulted in alkalization of the cytosol of B cells, CD4+ T cells, and CD8+ T cells as the result of Na+/H+ antiport activation. The change in intracellular Ca2+, production of IP3, and cytoplasmic alkalization in lymphocytes were due to its proteolytic activity. Cathepsin G released from granulocytes is considered to act on human lymphocytes in vivo and lead to the increase in DNA synthesis of B cells and CD4+ T cells through IP3 production, an increase in [Ca2+]i, and alkalization. However, these second messengers do not lead to the increase in DNA synthesis of CD8+ T cells.

Topics: Amino Acid Sequence; B-Lymphocytes; Calcium; Cathepsin G; Cathepsins; Cells, Cultured; Fura-2; Humans; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Lymphocyte Activation; Molecular Sequence Data; Serine Endopeptidases; T-Lymphocytes; Terpenes; Thapsigargin

Insulin may decrease the contractile response of vascular smooth muscle to vasoactive agents. This could be due to interactions of insulin with the effects of vasoactive agonists on intracellular free calcium transients in vascular smooth muscle cells. This study assesses the effects of physiological doses of insulin (70 microU/mL) on calcium responses in cultured vascular smooth muscle cells (primary unpassaged and passaged) to angiotensin II (1 nmol/L), arginine vasopressin (10 nmol/L), and norepinephrine (10 mumol/L). Intracellular free Ca2+ concentrations in single cells were measured microphotometrically using fura 2-AM. Insulin, angiotensin II, arginine vasopressin, and norepinephrine significantly increased calcium (to 115 +/- 7, 183 +/- 20, 184 +/- 15, and 168 +/- 12 nmol/L, respectively, from basal calcium of 90 +/- 10 nmol/L). Insulin significantly attenuated the agonist-induced calcium responses. The effects of insulin were almost completely inhibited by diltiazem, staurosporine, calphostin C, and thapsigargin. In conclusion, insulin stimulates calcium transients but blunts agonist-mediated calcium rises in vascular smooth muscle cells. These responses are related to regulatory effects of insulin on cellular calcium homeostasis and may explain how insulin modulates vascular smooth muscle contraction.

Topics: Alkaloids; Analysis of Variance; Angiotensin II; Animals; Arginine Vasopressin; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Diltiazem; Fluorescent Dyes; Fura-2; Humans; Insulin; Mesenteric Arteries; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Polycyclic Compounds; Protein Kinase C; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Staurosporine; Terpenes; Thapsigargin

Stimulation of neuropeptide Y (NPY) Y2 receptors induced an intracellular free Ca2+ ([Ca2+]i) increase in a human neuroblastoma cell line, CHP-234. When NPY in a Ca(2+)-free solution was applied, this increase was abolished. Depolarization with high KCl evoked no response, suggesting that the responses were not mediated by voltage-gated Ca2+ channels. There was no evidence that the NPY response consisted of a capacitative Ca2+ entry sensitive to internal Ca2+ store levels. The [Ca2+]i elevation was diminished by Ni2+, a blocker of Ca2+ entry. Mn2+ induced a quench of the fura-2 fluorescence, which ceased promptly upon the removal of NPY, indicating that Ca2+ entry was linked tightly to receptor activation. Although thapsigargin- and ryanodine-sensitive Ca2+ stores were present, NPY-induced responses were not impaired by pretreatment with either drug. Furthermore, NPY had no effect on the thapsigargin-sensitive store. Pertussis toxin did not affect the NPY-stimulated [Ca2+]i increase, although it abolished the NPY-dependent inhibition of cAMP production. It is concluded that the Y2 receptors couple directly to receptor-operated Ca2+ channels without the involvement of intracellular Ca2+ stores. The results also indicate that Y2 receptors can activate both pertussis toxin-sensitive and -insensitive mechanisms in the same cell.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Cell Line; Cyclic AMP; Fluorescent Dyes; Fura-2; Humans; Kinetics; Microscopy, Fluorescence; Neuroblastoma; Neuropeptide Y; Nickel; Pertussis Toxin; Potassium Chloride; Receptors, Neuropeptide Y; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.

Topics: Animals; Calcium; Cell Nucleus; Cytoplasm; Egtazic Acid; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Kinetics; Methacholine Chloride; Microscopy, Electron; Microscopy, Fluorescence; Mitochondria; Nuclear Envelope; Pancreatic Neoplasms; Rats; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channel Blockers; Cell Line; Cell Membrane; Fluorescent Dyes; Fura-2; Glioma; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Nimodipine; omega-Conotoxins; PC12 Cells; Peptides, Cyclic; Terpenes; Thapsigargin