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At least 1,063 records · Page 59Linked to original sources
PubMed1998

Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors.

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.

Animals↗
PubMed2005

Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism.

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.

Animals↗
PubMed1994

Early agonist-induced intracellular acidification is increased in platelets from patients with essential hypertension.

Enhanced Na+/H+ exchange has been reported to be increased in patients with essential hypertension. However, early variations of intracellular pH, although influenced by the antiport, are only partially dependent on the exchange. In this study, we measured the initial platelet pH response to agonists in a group of untreated subjects with essential hypertension (EH, n = 24) and in a group of age- and sex-matched normotensive control subjects (CS, n = 24). Intracellular pH was measured with the specific fluorescence indicator 2'7'bis(carboxyethyl)-5,6-carboxyfluorescein. Measurements were performed on platelets in the presence or absence of extracellular calcium, in a carbonate-free medium. Intracellular calcium was measured by the Fura 2 method. Mean pH values were slightly higher in the platelets of EH (7.469 +/- 0.017 U) compared with CS (7.423 +/- 0.012 U, P < .05), although there was a substantial overlap. When stimulated with physiologic agonists ADP and thrombin and with the calcium ionophore ionomycin, a biphasic response consisting of early acidification followed by alkalinization was observed, the second phase not being detectable with ADP. The initial acidification was greater in EH, particularly with ADP (EH, -0.046 +/- 0.002 U; CS, -0.036 +/- 0.002 U, P < .001) and with ionomycin (EH, -0.074 +/- 0.007 U; CS, -0.051 +/- 0.005 U, P < .05). This acidification proved in some way calcium dependent, as it was reduced in the absence of extracellular calcium (EGTA) in both EH and CS. After incubation with amiloride a further decrease in intracellular pH, more marked in EH, was observed. Alkalinization induced by thrombin was increased in EH (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1988

Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells.

Differentiation of cultured keratinocytes is regulated by the Ca2+ concentration of the culture medium. Below 0.1 mM Ca2+, a monolayer of basal cells is formed which fully differentiates in response to a rise in medium Ca2+. A role for protein kinase C in this differentiation program has been suggested because phorbol esters induce epidermal differentiation in cells grown in reduced Ca2+ medium, and exogenously added phospholipase C (which increases cellular diacylglycerol) mimics phorbol ester action. These findings suggested that the external Ca2+ signal may lead to protein kinase C activation via stimulation of cellular phospholipase C activity. The effect of the external Ca2+ signal on phospholipase C was studied in cultures prelabeled with [3H]-inositol. Within 2 min after addition of Ca2+ to 1 mM, an increase in inositol phosphates was measured. This correlated with a decrease in radiolabeled phosphoinositides, suggesting that these were the source of the increased inositol phosphates. After 3 h in 1 mM Ca2+ medium, each of the inositol phosphates remained increased to 130-140% of control levels. Inositol phosphate metabolism in neoplastic epidermal cells was quantitatively similar to normal cells in response to the Ca2+ signal. Stimulation of phosphatidylinositol (PIP) metabolism appears to be mediated by a rise in intracellular free Ca2+ because Ca2+ ionophores A23187 and ionomycin also cause a similar rise in inositol phosphate levels. Phorbol esters did not increase PIP turnover but instead stimulated phosphatidylcholine metabolism. The induction of epidermal differentiation by phorbol esters was enhanced by ionomycin, suggesting that both protein kinase C activation, elevation of intracellular calcium and PIP turnover were important components of the signal for epidermal differentiation. These results demonstrate that the second messenger system for Ca2+-mediated keratinocyte differentiation may be through a direct effect on phospholipase C activity.

Animals↗
PubMed1995

Age-related changes in cardiac norepinephrine release: role of calcium movement.

The purpose of this study was to determine if the age-related decrease in norepinephrine (NE) release from cardiac adrenergic nerve terminals is due to a defect in Ca2+ movement into the nerve terminal or to an alteration in Ca2+ activation of intracellular events leading to NE release. NE release was assessed in cardiac synaptosomes prepared from 6- and 24-month-old male F344 rats. K(+)-induced NE release was significantly greater in young vs old rats. Raising extracellular [Ca2+] increased NE release, but NE release always remained higher in the younger animals. Ionomycin, a Ca2+ ionophore, induced NE release from cardiac synaptosomes, and there was no age difference in the response. The age-related reduction in NE release induced by K+ and the capacity of ionomycin to induce similar NE release in young and old cardiac synaptosomes points to a reduction in Ca2+ movement during depolarization.

Aging↗
PubMed1995

Diminished cell-cell binding by lymphocytes from healthy, elderly humans: evidence for altered activation of LFA-1 function with age.

We studied the kinetics of cell-cell adhesion by monocyte-depleted peripheral blood lymphocytes with a stirred-cuvette aggregometer using samples from healthy young (mean age = 25 years) and healthy elderly (mean age = 75 years) human donors. This was a very reproducible assay, as there was virtually no intraindividual variability and very little interindividual variability among donors of the same age group. In all cases, aggregation of cells began immediately upon addition of the protein kinase C activator phorbol myristate acetate (PMA) or the calcium ionophore ionomycin, and continued until reaching an asymptotic limit by 20 minutes or less. Unstimulated cells did not aggregate during this time. The rate of aggregate formation and total amount of aggregation (using young donors' cells) varied between PMA- and ionomycin-stimulated cells, suggesting different mechanisms for initiating cellular aggregation, or possibly, different peripheral blood lymphocyte subsets affected by these activating agents. However, for both stimuli, the rate of aggregation and the total amount of cellular aggregation were significantly lower with the elderly donors' cells. Further, aggregation was Ca2+/Mg2+ dependent, and the reaction required metabolically active cells, as the reaction was inhibited by the addition of sodium azide and 2-deoxy-D-glucose in both donor groups. Pretreating cells with the actin polymerization inhibitor cytochalasin B resulted in 35-40% inhibition of aggregation among young donors' cells, although, interestingly, there was no apparent effect upon the cells capable of forming aggregates in the old donor group. In both donor groups, aggregation by activated cells could be partially blocked by pretreating cells with monoclonal antibodies directed against the alpha and beta chains of the integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18). These results show that there is diminished cell-cell binding among lymphocytes from healthy, elderly humans, and this is partly due to altered activation of LFA-1 function with age.

Adult↗
PubMed1998

Biochemical and kinetic characterization of the glucocorticoid-induced apoptosis of immature CD4+CD8+ thymocytes.

We characterized kinetic and biochemical changes during glucocorticoid (GC)-induced apoptosis of immature CD8+CD4+ double-positive (DP) thymocytes. A GC analog dexamethasone (Dex) induced rapid apoptotic commitment and a transient up-regulation of the NF-kappaB/RelA-p50-binding activity in DP cells. This required an early activation of proteasome, as judged by the ability of a specific proteasomal inhibitor, lactacystine, to delay apoptosis and to suppress Dex-dependent NF-kappaB activation. Dex-induced apoptotic commitment was preceded by the rapid (3 h) cleavage of both a typical caspase substrate, poly(ADP-ribose) polymerase (PARP), and of nuclear transcription factors AP-1, NF-kappaB p50-p50 and NUR-77. By contrast, phorbol myristate acetate (PMA) and/or ionomycin-induced apoptosis had much slower kinetics, were preceded by an early increase of NF-kappaB/RelA-p50, AP-1 and NUR-77 activities, and were insensitive to proteasome inhibition. Both the transgenic Bcl-2 and zVAD-fmk, an inhibitor of caspases, affected all features of Dex-induced apoptosis in a similar fashion, by inhibiting cell death and PARP cleavage, and by stabilizing AP-1, NF-kappaB p50-p50 and NUR-77 levels. Furthermore, Bcl-2 prevented Dex-induced RelA-p50 activation. However, a higher gene dosage of the transgenic Bcl-2 was required for protection against Dex, compared to the PMA and/or ionomycin-induced apoptosis. These findings highlight the unique mechanistic features of GC-induced apoptosis.

Acetylcysteine↗
PubMed1998

Analysis of the role of MKK-4/Sek-1 in T cell development and apoptosis.

The stress-activated protein kinases (SAPK) are a group of dual-specificity kinases with potential roles in the control of apoptosis and proliferation. In most cells they are regulated through phosphorylation by MKK-4. We have investigated the role of MKK-4 in T cell development and function by generating transgenic animals expressing catalytically inactive MKK-4 (dMKK-4) in the thymus. Our results show that overexpression of dMKK-4 does not interfere with normal T cell development. Furthermore, expression of dMKK-4 inhibits Fas- but not phorbol ester plus ionomycin-induced activation of SAPK, suggesting that a SAPK kinase different from MKK-4 is responsible for the regulation of SAPK activation after stimulation of T cells with phorbol ester plus ionomycin. We then analyzed the effect of dMKK-4 on Fas-induced apoptosis of thymocytes. Our results show that activation of SAPK is not a necessary event in Fas-induced apoptosis of thymocytes.

Animals↗
PubMed1995

Regulation of IFN-gamma mRNA production in murine natural killer cells.

We have previously reported that large, presumably in vivo activated, B cells stimulate murine natural killer (NK) cells to secrete increased levels of IFN-gamma. In order to further understand the mechanism of IFN-gamma induction, we compared the regulation of IFN-gamma mRNA production after stimulation of NK cells with either B lymphocytes or phorbol myristate acetate (PMA)+ionomycin. Here we show that stimulation of NK cells by either stimuli results increase in IFN-gamma mRNA, albeit with different kinetics. Although the induction requires new RNA synthesis, we could not detect increased transcription of the IFN-gamma gene after stimulation. Measurement of the rate of mRNA degradation after IFN-gamma mRNA has accumulated demonstrates that this mRNA is more stable than IFN-gamma mRNA from unstimulated NK cells. Together, these results suggest that the increase in IFN-gamma mRNA and protein in NK cells, stimulated by B cells or PMA+ionomycin, results from stabilization of pre-existing IFN-gamma message. Our results also suggest that induction of the factor which stabilizes the mRNA, although as yet unknown, requires new RNA synthesis.

Animals↗
PubMed2004

Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes.

We have shown that tumor vaccine-sensitized draining lymph node (vDLN) cells activated ex vivo with bryostatin and ionomycin (B/I) were capable of inducing antigen-specific regression of a murine mammary tumor, 4T07. vDLN cells not activated with B/I were ineffective. We hypothesized that B/I selectively activates tumor-sensitized (CD62Llow) lymphocytes, to account for the highly potent and tumor-specific activity. We hypothesized that CD8+ CD62Llow cells may be preferentially activated by B/I treatment, infiltrate the tumors and mediate tumor regression in mice. 4T07-IL2 tumor cells were injected into one hind footpad of BALB/c mice. Ten days later, vDLN were harvested and separated based on CD62L expression. After separation, cells were activated with B/I, expanded with IL2 (40 IU/ml) for 10 days, and adoptively transferred to 4T07 tumor bearing mice. Naive mice were also treated with different subsets of T cells and later were challenged with 4T07 tumor cells. To test in vitro responses to antigen, expanded lymphocytes were cultured either alone or with irradiated 4T07 tumor cells. Supernatants were harvested after 24 h and tested by ELISA for IFN-gamma. The importance of the host immune response was tested by AIT into 4T07-bearing nude athymic mice. Host mice were depleted in vivo of CD4 or CD8 T cells after vDLN AIT to ascertain the mediators of tumor regression. In order to track B/I activated vDLN cells, they were prestained with CFSE prior to adoptive transfer into tumor-bearing hosts. At various time points, tumors, spleens and lymph nodes of host mice were harvested, dual stained for activation marker expression and analyzed by flow cytometry. CD62Llow cells expanded 12-fold more than CD62Lhigh lymphocytes during the 10 day culture period. Supernatant from CD62Llow cells + 4T07 cultures contained 33-fold more IFN-gamma than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml +/- 135.8 vs 25.89 pg/ml +/- 0.01). Adoptive transfer of CD62Llow lymphocytes induced complete tumor regressions in all mice, while tumors regressed in only 17% of mice treated with CD62Lhigh lymphocytes. Naive mice that received B/I-activated CD62Llow cells were protected from future tumor challenges, while mice given CD62Lhigh cells did not exhibit the same resistance to tumor growth. Tumors in nude host mice regressed after AIT treatment. In vivo depletion of CD4 T cells after AIT did not inhibit tumor regression, but CD8 T cell depletion abrogated tumor regression. vDLN cells tracked preferentially to tumor draining lymph nodes and proliferated in vivo, persisting for at least 21 days, and were 95% CD44+ and 39% CD69+. Bryostatin 1 and ionomycin, by increasing PKC activity and intracellular calcium, respectively, mimic intracellular signals that result in T cell activation. CD62Llow cells are preferentially activated by B/I, leading to a highly effective anti-tumor T cell population.

Animals↗
PubMed1996

Importance of the phospholipase D-initiated sequential pathway for arachidonic acid release and prostaglandin D2 generation by rat peritoneal mast cells.

The association of prostaglandin D2 (PGD2) production as well as arachidonic acid release with the phospholipase D (PLD)-linked mechanism was studied in rat peritoneal mast cells. Stimulation of mast cells with cross-linking of the high-affinity Fc receptor for IgE caused increases in the release of arachidonic acid and PGD2, which are suppressed almost completely by ethanol or RHC 80267, a diacylglycerol lipase inhibitor. Ethanol did not influence inositol phosphate release in response to an antigen. An increase in diacylglycerol, that is inhibited by propranolol, was observed, with a peak within 1 min. Antigen stimulation induced little production of lysophosphatidylcholine, while ionomycin as a control markedly induced the production. However, the phospholipase A2 (PLA2) activity in the cytosol of antigen-stimulated cells increased to the level in ionomycin-stimulated cells. The addition of the ADP-ribosylation factor-containing fraction prepared from bovine brain, that is known to specifically activate PLD, to permeabilized mast cells in the presence of GTP gamma S, apparently increased arachidonic acid and PGD2 release, but not in the presence of ethanol. Furthermore, arachidonic acid release by an antigen was enhanced by melittin, that activates PLA2, but PGD2 production was not. These results suggest that antigen-stimulated PGD2 production as well as arachidonic acid release are strongly associated with the sequential PLD-linked pathway.

ADP-Ribosylation Factors↗
PubMed1995

Conformational aberrance of the sendai virus F0 protein in thapsigargin-treated cells allowing exit from the endoplasmic reticulum but causing arrest at the Golgi complex.

Thapsigargin and ionomycin inhibited the intracellular transport of the Sendai virus F0 protein. Depletion of Ca2+ from intracellular Ca2+ store(s) is critical for the inhibition, since ionomycin was more effective in the absence of extracellular Ca2+ than in its presence. Transport of F0 was arrested between the trans-Golgi complex and the plasma membrane [Ono, A. and Kawakita, M. (1994) J. Biochem. 116, 649-656], but only when thapsigargin was added before the synthesis of F0. This implies that F0 was committed to later arrest in the endoplasmic reticulum. Non-reducing SDS-polyacrylamide gel electrophoresis revealed a conformational abnormality of F0 immediately after pulse-labeling in thapsigargin-treated cells. The abnormality did not affect the exit of F0 from the endoplasmic reticulum, but paralleled its later arrest at the trans-Golgi stage. Pulse-labeled and 1-h-chased F0 was endoglycosidase H-resistant even in thapsigargin-treated cells, but was not recognized by mAb f-49, a monoclonal antibody that recognizes the corresponding F0 intermediate in uninhibited cells. The misfolded F0 may escape from recognition by means of the quality control system of the endoplasmic reticulum, but another system in the Golgi complex may complement the former. The arrested F0 was rapidly degraded.

3T3 Cells↗
PubMed1983

Factors affecting successful in vitro fertilization of bovine follicular oocytes.

These experiments were designed to define and optimize the efficiency of a system whereby bovine oocytes could be fertilized in vitro. The frequency of ova penetrated and the stage of fertilization were the end points examined. All experiments utilized cumulus-oocyte complexes from 1- to 5-mm follicles which were matured in vitro prior to fertilization. The experiments were designed to examine the effects of the following factors on fertilization: 1) pretreatment of sperm with ionomycin (a Ca++ ionophore), 2) preincubation of sperm at a high concentration and the presence of hypotaurine and epinephrine during fertilization, 3) the use of either follicle-stimulating hormone (FSH) or cAMP for the induction of cumulus expansion prior to fertilization, and 4) the need for the presence of cumulus cells during fertilization. Sperm exposure to ionomycin or preincubation at high sperm concentrations was not necessary for fertilization. The presence of hypotaurine and epinephrine during fertilization improved (P less than 0.05) the quality of fertilization (i.e., higher frequencies of oocytes with both female and male pronuclei were observed). However, they did not increase the percentage of ova penetrated (P greater than 0.05). Fertilization frequencies were not different (P greater than 0.05) between oocytes with cumulus expansion induced by FSH or cAMP. However, the use of either treatment resulted in higher fertilization rates when compared to untreated controls (P less than 0.05). Finally, while the presence of cumulus cells was not necessary for penetration of ova, increased frequencies of ova with both male and female pronuclei were found when cumuli were present (P less than 0.05).

Animals↗
PubMed1992

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.

Adenylyl Cyclases↗
PubMed1998

Segregation of the pathways leading to cortical reaction and cell cycle activation in the rat egg.

At fertilization of the mammalian egg, resumption of the cell cycle and the cortical reaction are two events of egg activation, correlated with an increase in intracellular Ca2+ concentration and activation of protein kinase C. To evaluate the pathways leading to both events, rat eggs were parthenogenetically activated by the calcium ionophore ionomycin, or by the protein kinase C activators 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG). Cortical granule exudate was visualized by the lectin Lens culinaris and Texas Red streptavidin, using a confocal microscope. Resumption of meiosis was detected by Hoechst dye, and intracellular Ca2+ concentration by fura-2. Ionomycin triggered both a cortical reaction and resumption of meiosis, while chelation of intracellular Ca2+ rise by BAPTA-AM (1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester) revealed a segregation between these two events. A low Ca2+ transient (approximately 150 nM) induced a partial cortical reaction in half of the eggs, but the meiotic status was not affected. TPA triggered a cortical reaction with neither resumption of meiosis nor intracellular Ca2+ rise, while OAG induced both aspects of activation, as well as a significant intracellular Ca2+ rise. We conclude that in the cascade of events leading to egg activation, the initial Ca2+ rise is followed by a segregation in the pathway. A relatively low Ca2+ rise is sufficient to induce a partial cortical reaction. However, a higher level of Ca2+ is required to complete the cortical reaction and resumption of meiosis. The activation of the cell cycle is Ca2+-dependent, but protein kinase C-independent.

Animals↗
PubMed1998

Localization and regulation of ryanodine receptor in bovine oocytes.

We have previously reported that injection of ryanodine receptor agonists into mature bovine oocytes induces intracellular calcium release, indicative of the existence of ryanodine receptors. In this experiment, further evidence of the ryanodine receptor localization, and developmental regulation in bovine oocytes is presented. The possible physiological significance is also suggested. Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes. As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more intense and continuous, yet not comparable to that observed in the metaphase II oocytes. These modifications correlate with the intracellular calcium responsiveness to ryanodine. A 200-microM injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stage bovine oocytes, while peaked intracellular calcium transients are subsequently observed in the metaphase II-stage oocytes. However, no significant changes in the amplitude of intracellular calcium transients induced by 250 nM inositol 1,4,5-trisphosphate and 10 microM ionomycin were observed in oocytes at a comparable stage. Fertilization induced a significant decrease in ryanodine receptor signal; similar changes were also observed in oocytes injected with 200 microM ryanodine or incubated with 10 microM ionomycin. However, no changes in ryanodine signal were observed in oocytes injected with vehicle medium. Furthermore, injection of either ryanodine or inositol 1,4,5-trisphosphate induced subsequent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cellular changes following fertilization; stimulation of this receptor results in the activation of bovine oocytes, and it is likely that this receptor may play a role at fertilization.

Animals↗
PubMed1998

Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes.

This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.

Animals↗
PubMed1991

Stimulated rise in neuronal calcium is faster and greater in the nucleus than the cytosol.

Calcium is an important regulator of a variety of neuronal activities including gene expression. However, it is not clear how Ca2+ influx affects intracellular Ca2+ concentration [( Ca2+]i) in the nucleus. We have taken advantage of laser photometry, the Ca2(+)-sensitive dye Indo-1 that allows ratio imaging, and confocal microscopy to eliminate the influences of unequal cell geometry and dye distribution. We show that Ca2+ influx into sympathetic neurons causes a significantly greater and faster increase in [Ca2+]i in the nucleus than in the cytosol. The differential increase in nuclear [Ca2+]i was apparent when Ca2+ entered from the extracellular medium during K+ depolarization, ionomycin or acetylcholine treatment, and brief periods of electrical stimulation. When intracellular Ca2+ was mobilized by caffeine the rise in nuclear [Ca2+]i was again greater than in any other region of the neuron. The increased nuclear Ca2+ levels were uniform throughout the nucleus and not associated with the nuclear envelope. The differential rise in nuclear Ca2+ was eliminated by acridine orange binding to nucleic acids. Nonexcitable cells (astrocytes, oligodendrocytes, and fibroblasts) did not show differential distribution of Ca2+ after ionomycin treatment. These results support the idea that activity-dependent gene regulation in sympathetic neurons may be mediated by changes in Ca2+ concentration at the level of the chromatin material.

Animals↗