Ca<Superscript>2+</Superscript> release from intracellular stores of pig oocytes at different stages of growth

Ca²⁺ release from intracellular stores of pig oocytes was investigated using the Ca²⁺-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl blue (BCB) staining. The stained oocytes (BCB “+”) were determined as the ones that completed their growth, while the stainless ones (BCB “−”) were determined as those in the final stages of growth. In the BCB “+” and BCB “−” oocytes, prolactin, theophylline, GTP, and GDP cause Ca²⁺ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca²⁺, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2⁺. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca²⁺ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca²⁺ release from intracellular stores in growing and grown pig oocytes.     

Effect of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes

Effect of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes was investigated using a fluorescent dye chlortetracycline. It is shown that in progesterone treated oocytes prolactin in concentration 50 ng/ml inhibits Ca2+ exit from intracellular stores of pig oocytes. Theophylline exerts the effect on prolactin Ca2+ exit from intracellular stores of pig oocytes. Employment of protein kinase C inhibitor cancelled inhibitory effect of prolactin and theophylline on Ca2+ exit from intracellular stores of pig oocytes. Ca2+ exit from intracellular stores of pig oocytes caused a joint influence of prolactin and GDP, and that of theophylline and GTP. The influence of protein kinase C inhibitor cancelled the stimulating effect of prolactin and GDP on Ca2+ exit from intracellular stores of pig oocytes also did not render any influence on the action of theophylline and GTP. These data suggest the influence of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes.     

Peculiarities of theophylline and prolactin interaction and calcium fluctuation from intracellular stores of porcine oocytes in the presence of estradiol

Using a fluorescent dye chlortetracycline, a study was made of the effect of estradiol on the interaction of theophylline and prolactin in the course of Ca2+ exit from intracellular stores of pig oocytes, isolated from ovaries at the stage of follicle growth. It is shown that in the presence of estradiol, prolactin does not stimulate Ca2+ exit from intracellular stores of pig oocytes. The action of theophylline similarly does not stimulate Ca2+ exit. Unlike, a joint effect of theophylline and prolactin on pig oocytes in the presence estradiol stimulated Ca2+ exit from intracellular stores of pig oocytes. These data demonstrated the influence of estradiol on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes.     

Effect of estradiol on stimulated theophylline and prolactin Ca2+ exit from intracellular stores of pig oocytes

Effect of estradiol on stimulated theophylline and prolactin Ca2+ exit from intracellular stores of pig oocytes was investigated using fluorescent dye chlortetracycline. It was shown that in the presence of estradiol neithert theophylline nor prolactin stimulated Ca2+ exit from intracellular stores of oocytes. Unlike, the common action oftheophylline and prolactin, also in the presence of estradiol, stimulated Ca2+ exit from intracellular stores. Inhibition of protein kinase C inhibits Ca2+ exit from intracellular stores in common action of theophylline and prolactin. These data suggest an obvious influence of estradiol on Ca2+ exit from intracellular stores of pig oocytes stimulated by theophylline and prolactin.     

Effects of guanine nucleotides and protein kinase C on prolactin-stimulated release of Ca from intracellular stores of pig oocytes

The effects of guanine nucleotides and protein kinase C on prolactin-stimulated Ca2+ release from intracellular stores of pig oocytes were studied using the fluorescent dye chlorotetracycline. The effect of prolactin was related to the protein kinase C activation. Inhibition of protein kinase C stimulated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin in the presence of extracellular Ca2+ and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. In a Ca2+-free medium, prolactin did not stimulate Ca2+ release from intracellular stores of the oocytes treated with GDP in the presence of GDP. GTP inhibition of protein kinase C activated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. These data suggest the influence of guanine nucleotides and protein kinase C on calcium metabolism, stimulated by prolactin.     

The influence of guanine nucleotides and protein kinase C on Ca2+ from intracellular stores of pigs oocytes stimulated by theophylline and cyclic AMP

Effect of guanine nucleotides and protein kinase C on Ca2+ exit from intracellular stores of pig oocytes, stimulated by theophylline and dbcAMP, was investigated using fluorescent dye chlortetracycline. Effect of cAMP on Ca2+ exit from intracellular stores of pig oocytes was not associated with activation of protein kinase C. In calcium-free medium, cAMP does not stimulate Ca2+ exit from intracellular stores of pig oocytes treated with GDP. In the presence of GDP, inhibition of protein kinase C activates Ca2+ exit from intracellular stores of pig oocytes on the action of cAMP. These data suggest the existence of different effects of guanine nucleotides on Ca2+ exit from intracellular stores of pig oocytes stimulated by cAMP.     

Influence of ryanodine and inositol trisphosphate receptors inhibitions on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP

The influence of ryanodine and inositol triphosphate receptors inhibitors on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP was investigated using fluorescent dye chlortetracycline. Porcine oocytes were isolated from ovaries with yellow body. Ca2+ exit from intracellular stores of porcine oocytes activated by prolactin (5 and 50 ng/ml) in calcium free medium was decreased after treatment of oocytes by heparin (inhibitor of inositol triphosphate receptors) and was not changed after treatment of oocytes by ruthenium red (inhibitor of ryanodine receptors). Inhibition of protein kinase C did not affect on the Ca2+ exit stimulated by prolactin. GTP did not stimulate Ca2+ exit from intracellular stores of pig oocytes, and inhibitors of both calcium channels and proteinkinase C had no influence on this process. The joint action of prolactin and GTP did not result in additional Ca2+ exit from intracellular stores of oocytes after both pretreatment and untreatment by the inhibitor of protein kinase C. The data obtained testify to activation of IP3-sensitive receptors under effect of prolactin and in the absence of GTP influence on these receptors.     

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of prolactin stimulated pig oocytes

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of pig oocytes activated by prolactin was investigated, using the fluorescent dye chlortetracycline. In the presence of extracellular calcium, the inhibitor of protein kinase C Ro 31-8220 increased calcium exit from intracellular stores in pig oocytes after prolactin treatment. In calcium-free medium, Ro 31-8220 exerted effect on calcium release from intracellular stores. In calcium-free medium, prolactin did not stimulate calcium release from intracellular stores of oocytes in the presence of thimerosal, while in the presence of protein kinase C inhibitor, prolactin increased Ca2+ content from intracellular stores in such oocytes. These data suggest a direct involvement of protein kinase C in the processes of regulation of Ca2+ exit from intracellular stores of pig oocytes stimulated by prolactin.     

Effects of Guanine Nucleotides and Protein Kinase C on Prolactin-Stimulated Release of Ca<Superscript>2+</Superscript> from Intracellular Stores of Pig Oocytes

The effects of guanine nucleotides and protein kinase C on prolactin-stimulated Ca²⁺ release from intracellular stores of pig oocytes were studied using the fluorescent dye chlorotetracycline. The effect of prolactin was related to the protein kinase C activation. Inhibition of protein kinase C stimulated Ca²⁺ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin in the presence of extracellular Ca²⁺ and inhibited Ca²⁺ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. In a Ca²⁺-free medium, prolactin did not stimulate Ca²⁺ release from intracellular stores of the oocytes treated with GDP in the presence of GDP. GTP inhibition of protein kinase C activated Ca²⁺ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin and inhibited Ca²⁺ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. These data suggest the influence of guanine nucleotides and protein kinase C on calcium metabolism, stimulated by prolactin.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 199–204.Original Russian Text Copyright © 2005 by Denisenko, Kuzmina.     

Identification of signal transduction pathway in fresh and vitrified porcine oocytes

Signal transduction pathway under the influence of somatotropin have been identified basis on the analysis of Ca2+ release from intracellular stores of fresh and vitrified porcine oocytes using inhibitory analysis. Somatotropin and GTP individually stimulated Ca2+ release from intracellular stores. The joint action of somatotropin and GTP activated additional Ca2+ release from intracellular stores both in fresh and vitrified porcine oocytes. Treatment of the oocytes with inhibitor of protein kinase C caused no additional Ca2+ release from intracellular stores. Ca2+ release from intracellular stores stimulated by GTP was connected with phosphate hydrolysis. Moving between intracellular Ca2+ depots stimulated by GTP was not determined by phosphate hydrolysis. Inhibitor of protein kinase C and microtubules were involved in the interaction of various intracellular depots. The data obtained suggest that signal transduction pathway in porcine oocytes do not change after vitrification.     

Effect of estradiol on Ca<Superscript>2+</Superscript> release from intracellular stores in porcine oocytes stimulated by prolactin,theophylline, or guanosine triphosphate

The interaction between prolactin and theophylline as well as between prolactin and guanosine triphosphate during Ca²⁺ release from intracellular stores of estradiol-treated porcine oocytes isolated from the ovary at the stage of follicular growth were studied using fluorescent Ca²⁺-sensitive probe chlortetracycline. In the absence of estradiol, prolactin or theophylline induced Ca²⁺ release from intracellular stores; however, no increase in Ca²⁺ release was observed after their combined action. Conversely, Ca²⁺ release from intracellular stores increased only after the combined exposure to prolactin and theophylline in the presence of estradiol. In the absence of estradiol, guanosine triphosphate induced calcium release alone and together with prolactin. Protein kinase C regulated Ca²⁺ release from intracellular stores after the combined exposure to prolactin and theophylline only in the presence of estradiol; while the activation of protein kinase C required no estradiol during the combined exposure to prolactin and guanosine triphosphate. The data obtained indicate the effect of estradiol on Ca²⁺ release from intracellular stores after the combined exposure to prolactin and theophylline, while no such effect was observed after the combined exposure to prolactin and guanosine triphosphate.     

Dependence of Ca2+ release from intracellular stores on NADH and FAD levels in fertilized and unfertilized bovine oocytes

Relation between NADH and FAD concentrations and the quantity of calcium released from intracellular stores in fertilized and unfertilized bovine oocytes was investigated using luminescent analysis. Inhibition of Ca2+ exit from intracellular stores was detected in degenerative oocytes at metaphase II and 2-cell embryos. The intensity of both NADH and FAD fluorescence increased in 2-cell degenerated embryos, whereas the increase in only NADH fluorescence intensity occurred in degenerated oocytes at metaphase II stage. Degeneration exerted no influence on NADH fluorescence intensity or Ca2+ exit from intracellular stores, whereas a decreased FAD fluorescence intensity was noted in degenerated pronuclei. The obtained data testify that in degenerated zygotes and early embryos Ca2+ release may occur from different intracellular stores.     

Identification of signal transduction pathways in fresh and vitrified porcine oocytes

Pathways are identified of signal transduction upon the action of somatotropin on the basis of analysis of fluctuation of the calcium content in intracellular depots of native and devitrified pig oocytes with the use of inhibitor analysis. STH, as well as GTP, has been shown to stimulate Ca²⁺ release from intracellular depots; their combined action activates additional release of Ca²⁺ from intracellular depots both in native and in devitrified oocytes. Treatment of oocytes with the protein kinase C inhibitor did not cause additional release of Ca²⁺ from intracellular depots. The release from intracellular depots stimulated by Ca²⁺ is connected with phosphate hydrolysis. GTP-stimulated translocation of Ca²⁺ between intracellular depots was not determined by phosphate hydrolysis. Protein kinase C and microtubules are involved in interactions of various intracellular depots. The obtained data indicate that, after devitrification, the signal transduction pathways in oocytes are not submitted to changes.     

Effect of the protein kinase C inhibitor on changes in Ca(2+) level in pig granulosa cells induced by prolactin

The effect of the PKC inhibitor on Ca2+ responses to prolactin in the pig granulosa cells was studied using fluorescent dye and chlortetracycline. The effect was shown to be connected with activation of the PKC. The Ro 31-8220 increased penetration of extracellular calcium and exit of calcium from intracellular stores. The data obtained suggest an involvement of the PKC in changes of calcium contents in the pig granulosa cells activated by prolactin.     

Effect of guanosine triphosphate on the release of Ca2+ from intracellular store sites of saponin-treated human peripheral lymphocytes

The effects of guanosine triphosphate (GTP) on the release and uptake of Ca2+ in nonmitochondrial intracellular store sites of human peripheral lymphocytes were examined. GTP in the presence of 3% polyethylene glycol released Ca2+ from the intracellular store sites of lymphocytes in a dose-dependent manner, and the maximal release was obtained at 10 microM GTP. GDP and 5'-GMP also enhanced the release of Ca2+. On the other hand, Ca2+ uptake in the presence of oxalate by saponin-treated lymphocytes was stimulated by GTP and this stimulation was abolished when polyethylene glycol was concomitantly present. The dose dependence of the stimulated Ca2+ uptake by GTP was much the same as that of the Ca2+ released by GTP. These results indicate that GTP has an inherent activity to release Ca2+ as well as to stimulate the uptake of Ca2+ in nonmitochondrial intracellular store sites of saponin-treated lymphocytes. The stimulatory effect of polyethylene glycol on GTP-mediated Ca2+ release may occur by inhibiting functions of the Ca2+ pump.     

Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C

alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G-protein-coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha z, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca(2+)-dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2(+)-independent component of LTX-stimulated release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non-selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca2+ provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion.     

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

Effect of guanosine triphosphate on the release and uptake of Ca2+ in saponin-permeabilized macrophages and the skeletal-muscle sarcoplasmic reticulum.

The effects of GTP, with or without polyethylene glycol (PEG), on the release and uptake of Ca2+ were examined by using saponin-treated macrophages and sarcoplasmic reticulum isolated from skeletal muscles. The application of GTP in concentrations in the range 0.1-10 microM induced a gradual, small but sustained release of Ca2+ from the saponin-treated macrophages. The addition of PEG to GTP markedly enhanced the GTP-mediated Ca2+ release. GTP at the same concentration ranges used for Ca2+ release decreased the amount of Ca2+ uptake, at a steady state, but stimulated the rate of Ca2+ accumulation in the presence of oxalate, the Ca2+-precipitating anion. The addition of PEG abolished the GTP-evoked stimulation of Ca2+ accumulation in the presence of oxalate. The stimulating effect on the rate of Ca2+ accumulation by GTP and its elimination by PEG were not due to changes in the permeability of oxalate by either GTP or PEG, or both. The Ca2+-releasing effect of GTP without PEG was enhanced by eliminating the uptake activity by decreasing the content of ATP. These results indicate that GTP has an inherent activity to release Ca2+ from non-mitochondrial intracellular stores of saponin-treated macrophages, and PEG enhances the GTP-mediated Ca2+ release, partly owing to its eliminating effect on GTP-stimulated Ca2+ uptake activity. These effects of GTP observed with saponin-permeabilized macrophages were not apparent in the isolated skeletal-muscle sarcoplasmic reticulum.     

Agonist-mediated Ca2+ release in permeabilized UMR-106-01 cells. Transport properties and generation of inositol 1,4,5-trisphosphate

Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.     

Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR)

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will focus on the effects of cADPR in different cells and tissues, the mechanisms of cADPR-mediated Ca2+ release and Ca2+ entry, extracellular effects of cADPR, and the role of cADPR in a cell system studied in detail, human T-lymphocytes.