calcimycin and fura-2-am

Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of the present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5-7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K(+)-stimulated uptake of (45)Ca(2+) and [Ca(2+)](i), measured with the Ca(2+)-sensitive fluorescent dye fura-2AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell-permeable analog of cAMP, had no effect on inhibition of K(+)-stimulated (45)Ca(2+) influx or [Ca(2+)](i) by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K(+), which requires Ca(2+) influx through voltage-gated Ca(2+) channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca(2+)](i).

Topics: Analysis of Variance; Animals; Arylalkylamine N-Acetyltransferase; Calcimycin; Calcium; Cells, Cultured; Chick Embryo; Clozapine; Cyclic AMP; Cyclic GMP; Dopamine; Dopamine Antagonists; Dose-Response Relationship, Drug; Fura-2; GABA Antagonists; Intracellular Fluid; Ionophores; Potassium; Receptors, Dopamine D4; Retina; Retinal Cone Photoreceptor Cells; Spiperone

A fourth intracellular Ca2+ pool in Leishmania donovani was identified by permeabilizing plasma membrane with digitonin. In Fura 2 loaded cells Ca2+ was released synergistically when mitochondrial function was blocked by antimycin and oligomycin. Vanadate did not have any effect if applied before incorporation of these mitochondrial poisons. However, the same inhibitor which inhibits Ca2+-ATPase activity of endoplasmic reticulum was able to release Ca2+ at a slow rate when added after antimycin and oligomycin. Alkalization of cytoplasmic pH allowed further release of Ca2+ essentially from the acidocalcisome. Purified glycosomes could mediate Ca2+ uptake mechanism in presence of vanadate whereas bafilomycin, a specific and potent inhibitor of vacuolar proton pump did not have any effect. Glycosomal Ca2+-ATPase activity was optimum at pH 7.5. The apparent Km for calciumin presence of vanadate was 12 nM. Taken together, it may be suggested that a vanadate-insensitive Ca2+-ATPase is present in the membrane of this microbody. Presence of glycosomal Ca2+ was further confirmed by imaging of Ca2+ activity in the Fura 2 loaded purified organelle using confocal laser. Results reveal that newly localized glycosomal calcium may essentially be an effective candidate to play a significant role in cellular function.

Topics: Animals; Antimycin A; Calcimycin; Calcium; Calcium-Transporting ATPases; Digitonin; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Ionophores; Leishmania donovani; Macrolides; Microbodies; Microscopy, Confocal; Oligomycins; Spectrometry, Fluorescence; Uncoupling Agents; Vanadates

Endothelial cell (EC) migration, essential for reestablishing arterial integrity after vascular injury, is inhibited by oxidized LDL (oxLDL) and lysophosphatidylcholine (lysoPC) that are present in the arterial wall. We tested the hypothesis that a mechanism responsible for lysoPC-induced inhibition is increased intracellular free calcium concentration ([Ca(2+)](i)).. LysoPC, at concentrations that inhibit in vitro EC migration to 35% of control, increased [Ca(2+)](i) levels 3-fold. These effects of lysoPC were concentration dependent and reversible. LysoPC induced Ca(2+) influx within 10 minutes, and [Ca(2+)](i) remained elevated for 2 hours. The calcium ionophore A23187 also increased [Ca(2+)](i) and inhibited EC migration. Chelators of intracellular Ca(2+) (BAPTA/AM and EGTA/AM) and nonvoltage-sensitive channel blockers (lanthanum chloride and gadolinium chloride) blunted the lysoPC-induced [Ca(2+)](i) rise and partially preserved EC migration. After lysoPC treatment, calpain, a calcium-dependent cysteine protease, was activated, and cytoskeletal changes occurred. Calpain inhibitors (calpastatin, MDL28170, and calpeptin) added before lysoPC prevented cytoskeletal protein cleavage and preserved EC migration at 60% of control levels.. LysoPC increases [Ca(2+)](i). In turn, activating calpains that can alter the cytoskeleton are activated and EC migration is inhibited.

Topics: Animals; Aorta; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calpain; Cattle; Cell Movement; Chelating Agents; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Endothelium, Vascular; Enzyme Activation; Fluorescent Dyes; Fura-2; Ionophores; Lysophosphatidylcholines

1. It is well established that the activity of Na+,K+-ATPase (NKA) is regulated by protein kinases A (PKA) and C (PKC), but results on their effects have been conflicting. The aim of this study was to examine if this is ascribed to the intracellular concentration of Ca2+ ([Ca2+]i). 2. Rat renal NKA was stably expressed in COS cells (green monkey kidney cells). Increases in [Ca2+]i were achieved with the Ca2+ ionophore A23187 and verified by direct measurements of [Ca2+]i using fura-2 AM as an indicator. The activity of NKA was measured as ouabain-sensitive 86Rb+ uptake and the state of phosphorylation of NKA was monitored with two site-directed phosphorylation state-specific antibodies. 3. Activation of PKA with forskolin decreased NKA activity by 45.5 +/- 8.9 % at low [Ca2+]i (120 nM) and increased it by 40.5 +/- 6.4 % at high [Ca2+]i (420 nM). The change in NKA activity by forskolin correlated with the level of increase in [Ca2+]i. 4. The effect of 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a specific PKC activator, on the activity of NKA was also Ca2+ dependent, being inhibitory when [Ca2+]i was low (29.3 +/- 3.6 % decrease at 120 nM Ca2+) and stimulatory when [Ca2+]i was high (36.6 +/- 10.1 % increase at 420 nM Ca2+). 5. The alpha subunit of NKA was phosphorylated under both low and high [Ca2+]i conditions upon PKA or PKC activation. PKA phosphorylates Ser943. PKC phosphorylates Ser23. 6. To see if the observed effects on NKA activity are secondary to changes in Na+ entry, we measured NKA hydrolytic activity using permeabilized membranes isolated from cells under controlled Na+ conditions. A decreased activity at low [Ca2+]i and no change in activity at high [Ca2+]i were observed following forskolin or OAG treatment. 7. Purified NKA from rat renal cortex was phosphorylated and inhibited by PKC. This phosphorylation-associated inhibition of NKA was neither affected by Ca2+ nor by calmodulin, tested alone or together. 8. We conclude that effect of PKA/PKC on NKA activity is dependent on [Ca2+]i. This Ca2+ dependence may provide an explanation for the diversity of responses of NKA to activation of either PKA or PKC.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Cell Membrane; Colforsin; COS Cells; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Fluorescent Dyes; Fura-2; Kidney; Phosphorylation; Protein Kinase C; Rats; Rubidium Radioisotopes; Sodium-Potassium-Exchanging ATPase

The purpose of the present work was to characterize calcium responses of brain-capillary endothelial cells (BCEC), the cells forming the blood-brain barrier, to chemical, hyperosmolar and mechanical stimulation. Confluent BCEC cultures were grown from capillary fragments isolated from rat cerebral cortex. Intracellular free calcium ([Ca2+]i) was measured using fura-2 and digital imaging. Our experiments show large endothelial calcium responses to substance P and ATP, up to a peak value of approximately 1000 and 600 nM, respectively, and these responses were observed in 2/3 of the cells. Calcium responses to bradykinin, histamine, and hyperosmolar sucrose or mannitol were smaller, attaining a peak in the range 180-340 nM, and were observed in a smaller fraction of the cells. No calcium responses were observed to high-potassium, L-glutamate, serotonin, carbachol, noradrenaline, and nitric-oxide donors. Consecutive superfusion of the cultures with ATP, bradykinin, and histamine showed that cells with a certain response pattern were spatially grouped; the response pattern itself varied widely between experiments. Mechanical stimulation of a single cell caused a calcium response in the stimulated cell in primary cultures and triggered an intercellularly propagating calcium wave in passaged cultures. Given the important effect of endothelial [Ca2+]i on blood-brain barrier permeability and transport, we conclude that substance P and ATP are potential modulators of blood-brain barrier function. Hyperosmolarity-induced blood-brain barrier opening is probably not mediated through endothelial [Ca2+]i.

Topics: Adenosine Triphosphate; Animals; Bradykinin; Calcimycin; Calcium; Capillaries; Carbachol; Cells, Cultured; Cerebral Cortex; Endothelium, Vascular; Fluorescent Dyes; Fura-2; Histamine; Hypertonic Solutions; Norepinephrine; Rats; Rats, Wistar; Substance P; Thimerosal

There is increasing evidence that local substance P (SP) exacerbates peripheral inflammations, partly by stimulating production of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). SP may play similar roles in certain central nervous system inflammations. Multiple sclerosis plaques, for example, form around veins which are innervated by unmyelinated SP-containing fibers, and astrocytes in multiple sclerosis plaques stain for SP. We tested whether SP could stimulate IL-1 and TNF alpha production by cultured astrocytes and whether calcium was the second messenger in this process. We found that both SP and the calcium ionophore A23187 raised intracellular calcium ([Ca2+]i) and stimulated IL-1 production in astrocytes. SP also nonsignificantly increased TNF alpha production by astrocytes. Treatment with dibromo BAPTA/AM, an intracellular calcium buffer, blocked SP-induced IL-1 production. These findings indicate that SP induces IL-1 production by astrocytes and uses calcium as a second messenger. Our results indicate local SP may play a role in multiple sclerosis and certain other central nervous system inflammations.

Topics: Animals; Animals, Newborn; Astrocytes; Brain; Calcimycin; Calcium; Cells, Cultured; Egtazic Acid; Fluorescent Dyes; Fura-2; Immunohistochemistry; Interleukin-1; Kinetics; Lipopolysaccharides; Lipoproteins, LDL; Rats; Substance P; Tumor Necrosis Factor-alpha

Cytosolic free Ca2+ ion concentrations were ([Ca2+]i) measured in single swine granulosa cells using the Ca2(+)-sensitive fluorescent indicator dye fura-2 and digital imaging videomicroscopy with high spatial and temporal resolution. Ovine FSH (oFSH) elicited specific [Ca2+]i increases, which reached their highest value within 5 min of the onset of stimulation and were sustained for 10-15 min. [Ca2+]i usually returned to prestimulated levels and cells regained full responsiveness to a second exposure to oFSH within 20 min. The effect of FSH was not blocked by pretreatment with pertussis toxin. Forskolin and 8-bromo-cAMP were able to mimic similar increases in [Ca2+]i in the presence or absence of extracellular Ca2+. In contrast, the oFSH-induced [Ca2+]i rises were abolished in the absence of extracellular Ca2+. The Ca2+ channel blocker verapamil completely abolished the oFSH-induced [Ca2+]i rise, but not the 8-bromo-cAMP- or the forskolin-induced [Ca2+]i rise. In summary, we have demonstrated that [Ca2+]i is regulated by oFSH in single swine granulosa cells. We propose that the transducing pathway for this FSH effect might not involve cAMP, and that whichever second messenger is responsible for the Ca2+ signal generation acts upon plasma membrane Ca2+ channels.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcimycin; Calcium; Cells, Cultured; Cyclic AMP; Cytosol; Female; Fluorescent Dyes; Follicle Stimulating Hormone; Fura-2; Granulosa Cells; Kinetics; Spectrometry, Fluorescence; Swine