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.     

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.     

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.     

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 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.     

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.     

Ca2+ release from intracellular stores of pig oocytes during different stages of growth

Ca2+ release from intracellular stores of pig oocytes was investigated using the Ca(2+)-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl clue (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.     

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.     

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.     

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.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

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.     

Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons

Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.     

细胞内贮存钙释放的机制

细胞内贮存钙的释放主要由１,４,５－三磷酸肌醇（ＩＰ３）受体系统和ｒｙａｎｏｄｉｎｅ受体系统调控。前通过ＩＰ３与其受体结合后,诱发细胞内钙释放;后通过复杂的机制调节环腺苷二磷酸核糖含量,由ｃＡＤＰＲ直接或间接作用于ｒｙａｎｏｄｉｎｅ受体,进而启动由Ｃａ＾２＋诱发的Ｃａ＾２＋释放机制。上述两系统之间相互作用,共同调节细胞内贮存钙的释放。     

Activation of in vitro matured pig oocytes using activators of inositol triphosphate or ryanodine receptors

In our study, we observed the activation of in vitro matured pig oocytes and their subsequent parthenogenetic cleavage after stimulation of ryanodine receptors (RyR) using ryanodine (Ry), caffeine or cyclic adenosine diphosphate ribose (cADPri) or after stimulation of inositol triphosphate receptors (IP(3)R) using D-myo-inositol 1,4,5-triphosphate (IP(3)). Heparin, a potent blocker of IP(3)R, prevented the activation of porcine oocytes using IP(3), but blockers of RyR (ruthenium red or procaine) prevented activation after stimulation by RyR and stimulation by IP(3)R using IP(3). The drugs were injected into oocytes matured to the stage of metaphase II and activation was determined by assessment of pronuclear formation. The activity of H1 kinase was determined and our results demonstrated a significant drop in H1 activity in the activated oocytes. The cleavage of parthenogenetic embryos progresses to more advanced stages after stimulation by IP(3)R than after stimulation by RyR. Our results could indicate that, in pig oocytes, the calcium released from IP(3)-sensitive stores triggers the calcium release from ryanodine-sensitive intracellular stores, which is necessary for oocyte activation. The calmodulin inhibitors ophiobolin A and W7 reduce the activation of oocytes induced by stimulation of RyR or IP(3)R.     

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.     

Capacitative calcium entry mechanism in porcine oocytes

The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry. A similar divalent cation influx could also be detected with the Mn(2+)-quench technique after inositol 1,4,5-triphosphate-induced Ca(2+) release. In both cases, lanthanum, the Ca(2+) permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca(2+) influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca(2+) entry mechanism might help in refilling the intracellular stores after the release of Ca(2+) from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca(2+) entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca(2+) entry mechanism and thus contributes to Ca(2+) influx.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Cold-induced calcium elevation triggers DNA fragmentation in immature pig oocytes

Fluo-4 loaded immature oocytes were cooled from 30 degrees C to various lower temperatures between 20 and 10 degrees C and changes in intracellular calcium (Ca(2+)) levels were measured. Pig oocytes cooled to 14 degrees C exhibited a clear biphasic Ca(2+) rise. Lower temperatures produced similar responses, while higher temperatures did not exert any effect. The Ca(2+) response appeared to rely on ryanodine dependent stores as removal of extracellular Ca(2+) and intracytoplasmic injection of heparin did not modify cold-induced Ca(2+) elevation, while procaine or ruthenium red virtually eliminated the response. Confocal analysis of subcellular Ca(2+) distribution during cooling revealed that the ion rises sharply within the nucleus. As Ca(2+) imbalance may activate nuclear endonucleases, DNA integrity of cooled pig oocytes was evaluated by TUNEL and comet assays. Most cooled oocytes showed clear signs of DNA fragmentation. Oocytes injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetrapotassium salt (BAPTA), a Ca(2+) chelator, maintained their DNA integrity thus confirming that intracellular Ca(2+) is involved in triggering DNA fragmentation. The protective effect exerted by ruthenium red and/or procaine further confirmed this hypothesis. These data show that a moderate and transient cooling is sufficient to cause an intracellular Ca(2+) rise that leads to DNA damage. The addition of inhibitors of ryanodine dependent Ca(2+) stores may represent a valuable protective treatment to reduce chilling injuries.     

Desensitization of angiotensin II: effect on [Ca2+]i, inositol triphosphate, and prolactin in pituitary cells

Activation of pituitary angiotensin (ANG II) type 1 receptors (AT1) mobilizes intracellular Ca2+, resulting in increased prolactin secretion. We first assessed desensitization of AT1 receptors by testing ANG II-induced intracellular Ca2+ concentration ([Ca2+](i)) response in rat anterior pituitary cells. A period as short as 1 min with 10(-7) M ANG II was effective in producing desensitization (remaining response was 66.8 +/- 2.1% of nondesensitized cells). Desensitization was a concentration-related event (EC(50): 1.1 nM). Although partial recovery was obtained 15 min after removal of ANG II, full response could not be achieved even after 4 h (77.6 +/- 2.4%). Experiments with 5 x 10(-7) M ionomycin indicated that intracellular Ca2+ stores of desensitized cells had already recovered when desensitization was still significant. The thyrotropin-releasing hormone (TRH)-induced intracellular Ca2+ peak was attenuated in the ANG II-pretreated group. ANG II pretreatment also desensitized ANG II- and TRH-induced inositol phosphate generation (72.8 +/- 3.5 and 69.6 +/- 6.1%, respectively, for inositol triphosphate) and prolactin secretion (53.4 +/- 2.3 and 65.1 +/- 7.2%), effects independent of PKC activation. We conclude that, in pituitary cells, inositol triphosphate formation, [Ca2+](i) mobilization, and prolactin release in response to ANG II undergo rapid, long-lasting, homologous and heterologous desensitization.