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.     

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.     

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<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.     

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.     

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.     

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.     

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.     

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.     

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.     

Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells

The role of guanine nucleotides in insulin secretion was investigated in electrically permeabilized RINm5F cells. Ca2+ stimulated insulin release (EC50 approximately 2 microM Ca2+). The GTP stable analog, GTP gamma S, elicited insulin secretion at vanishingly low Ca2+ concentrations (less than 10(-11) M), slightly potentiated the response to intermediate Ca2+ levels, but exerted less than additive effects at maximal Ca2+ concentrations. The GDP analog, GDP beta S, inhibited both GTP gamma S- and Ca2+-stimulated secretion. The action of GTP gamma S was not mediated by cAMP, as the latter only enhanced Ca2+-induced secretion. In contrast, 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted insulin release at nonstimulatory Ca2+ levels as well as potentiating the Ca2+ response. GTP analogs stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), as assessed by inositol phosphate generation. However, this could not fully explain guanine nucleotide-induced secretion because: GTP gamma S-stimulated PtdInsP2 breakdown was totally dependent on Ca2+ and abolished at Ca2+ below 10(-11) M; at these Ca2+ levels, activators of protein kinase C were weak or ineffective secretagogues; the GTP analog Gpp(NH)p was much less effective than GTP gamma S in activating PtdInsP2 hydrolysis, while fully mimicking the effect on Ca2+-independent secretion. Both GTP gamma S-induced PtdInsP2 hydrolysis and insulin release were insensitive to pertussis toxin and cholera toxin. The findings point to a guanine nucleotide-regulated site in the activation of insulin secretion different from the known transmembrane signalling systems.     

Short-term plasticity of cyclic adenosine 3',5'-monophosphate signaling in anterior pituitary corticotrope cells: the role of adenylyl cyclase isotypes

Anterior pituitary corticotropes show a wide repertory of responses to hypothalamic neuropeptides and adrenal corticosteroids. The hypothesis that plasticity of the cAMP signaling system underlies this adaptive versatility was investigated. In dispersed rat anterior pituitary cells, depletion of intracellular Ca2+ stores with thapsigargin combined with ryanodine or caffeine enhanced the corticotropin releasing-factor (CRF)-evoked cAMP response by 4-fold, whereas reduction of Ca2+ entry alone had no effect. CRF-induced cAMP was amplified 15-fold by arginine-vasopressin (AVP) or phorbol-dibutyrate ester. In the presence of inhibitors of cyclic nucleotide phosphodiesterases and phorbol-dibutyrate ester, the depletion of Ca2+ stores had no further effect on CRF-induced cAMP accumulation. Adenohypophysial expression of mRNAs for the Ca2+-inhibited adenylyl cyclases (ACs) VI and IX, and the protein kinase C-stimulated ACs II and VII was demonstrated. ACIX was detected in corticotropes by immunocytochemistry, whereas ACII and ACVI were not present. The data show negative feedback regulation of CRF-induced cAMP levels by Ca2+ derived from ryanodine receptor-operated intracellular stores. Stimulation of protein kinase C by AVP enhances Ca2+-independent cAMP synthesis, thus changing the characteristics of intracellular Ca2+ feedback. It is proposed that the modulation of intracellular Ca2+ feedback in corticotropes by AVP is an important element of physiological control.     

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.     

Mitogenesis of 3T3 fibroblasts induced by endogenous ganglioside is not mediated by cAMP, protein kinase C, or phosphoinositides turnover

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.     

Ca2(+)-dependent amylase secretion from pancreatic acinar cells occurs without activation of phospholipase C linked G-proteins

We have examined the influence of guanine nucleotides on Ca2(+)-dependent amylase secretion from SLO permeabilized rat pancreatic acini. GTP gamma S (100 microM) stimulated Ca2+ dependent amylase release, decreasing the EC50 for Ca2+ from 1.4 to 0.8 microM. By contrast, GDP (1mM) and dGDP (1mM) inhibited the maximal Ca2(+)-dependent secretory response. Measurement of IP3 liberation showed that Ca2+ stimulation did not increase the activity of phospholipase C (PLC) postulated to be linked to a G-protein termed Gp; GDP and dGDP must therefore be exerting their inhibitory action via a GTP-binding protein distinct from the PLC-linked Gp.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3',5'-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways.

The cDNAs encoding the murine LH receptor (LHR) and the human beta 2-adrenoceptor (h beta 2AR) were cloned and RNAs complementary to their sense strands (cRNAs) were injected into defolliculated Xenopus oocytes. This led to expression, respectively, of LH- and isoproterenol-stimulable adenylyl cyclase activities, indicating that functionally active receptor cDNAs had been cloned. In oocytes injected with LHR cRNA, but not in control or h beta 2AR cRNA-injected oocytes, human CG and LH increased a Ca(2+)-activated Cl- current, as measured by the two-microelectrode voltage-clamp method. This effect was not seen with isoproterenol in control or h beta 2AR cRNA-injected oocytes, it was also not observed in response to forskolin or (Bu)2cAMP. The response to human CG could be obtained in the absence of extracellular Ca2+ but was abolished by injection of EGTA, indicating that it was caused by mobilization of Ca2+ from intracellular stores. The response was unaffected by overnight treatment with 1 microgram/ml pertussis toxin. The experiments show that a glycoprotein hormone receptor can be expressed as a functionally active molecule in Xenopus oocytes, and that the LHR has the ability of activating two separate intracellular signaling pathways: one forming the second messenger cAMP, and the other mobilizing Ca2+ from intracellular stores. It is proposed that the latter is secondary to a primary activation of phospholipase C by the LHR, which elevates intracellular Ca2+ via intermediary elevation of inositol phosphates, presumably (1,4,5)inositol trisphosphate.     

NMDA-receptor regulation of muscarinic-receptor stimulated inositol 1,4,5-trisphosphate production and protein kinase C activation in single cerebellar granule neurons

Inositol 1,4,5-trisphosphate (InsP(3)) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C delta1 tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta1)). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+-release. Thus, methacholine (MCH), acting via muscarinic acetylcholine-receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo-3 and fura-4f, but not fura-2. Confocal measurements of single CGNs expressing eGFP-PH(PLCdelta1) demonstrated that MCH stimulated a robust peak increase in InsP(3), which was followed by a sustained plateau phase of InsP(3) production. In contrast, glutamate-induced InsP(3) signals were weak or not detectable. MCH-stimulated InsP(3) production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA- and KCl-mediated Ca2+-entry significantly enhanced MCH-induced InsP(3) production. Furthermore, mAchR-mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 microm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA-mediated Ca2+ influx and PLC activation may represent a coincident-detection system whereby ionotropic and metabotropic signals combine to stimulate InsP(3) production and PKC-mediated phosphorylation events in CGNs.     

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.     

A Novel Effect of Cyclic AMP on Capacitative Ca2+ Entry in Cultured Rat Cerebellar Astrocytes

One of the most important intracellular Ca2+ regulatory mechanisms in nonexcitable cells, "capacitative Ca2+ entry" (CCE), has not been adequately studied in astrocytes. We therefore investigated whether CCE exists in cultured rat cerebellar astrocytes and studied the roles of cyclic AMP (cAMP) and protein kinase C (PKC) in CCE. We found that (1) at least two different intracellular Ca2+ stores, the endoplasmic reticulum and mitochondria, are present in cerebellar astrocytes; (2) CCE does exist in these cells and can be inhibited by Ni2+, miconazole, and SKF 96365; (3) CCE can be directly enhanced by an increase in intracellular cAMP, as 8-bromoadenosine 3',5'-cyclic monophosphate (8-brcAMP), forskolin, and isobutylmethylxanthine have stimulatory effects on CCE; and (4) neither of the two potent protein kinase A (PKA) inhibitors, H8 and H89, nor a specific PKA agonist, Sp-adenosine 3',5'-cyclic monophosphothioate, had a significant effect on cAMP-enhanced Ca2+ entry. The [Ca2+]i increase was not due to a release from calcium stores, hyperpolarization of the membrane potential, inhibition of calcium extrusion, or a change in pHi, suggesting that cAMP itself probably acts as a novel messenger to modulate CCE. We also conclude that activation of PKC results in an increase in CCE. cAMP and PKC seem to modulate CCE by different pathways.