Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.
(9/3227)
The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster (approximately 70 kDa, pI 5) and a lower molecular weight (approximately 35-45 kDa, pI 5.5) protein cluster were released from the oolemmal surface as a result of PI-PLC treatment. It is likely that these GPI-anchored egg surface proteins are required for sperm-egg binding and fusion. (+info)
Inhibition of copper/zinc superoxide dismutase impairs NO.-mediated endothelium-dependent relaxations.
(10/3227)
The superoxide anion (O-2.) appears to be an important modulator of nitric oxide (NO.) bioavailability. The present study was designed to characterize the role of copper/zinc superoxide dismutase (Cu/Zn SOD) in endothelium-dependent relaxations. Cu/Zn SOD was inhibited with the Cu2+ chelator diethyldithiocarbamic acid (DETCA). In isolated canine basilar arteries, DETCA (7.6 x 10(-3) M) inhibited total vascular SOD activity by 46% (P < 0.0001, n = 6-8 dogs). DETCA (7.6 x 10(-3) M) significantly reduced relaxations to bradykinin and A-23187 (P < 0.05, n = 7-11). The inhibitory effect of DETCA was abolished by the O-2. scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron; 9.4 x 10(-3) M; P < 0.05, n = 6-13). Tiron significantly potentiated the relaxations to bradykinin in control rings (P < 0.05, n = 13), and the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M) abolished these relaxations (P < 0.0001, n = 6). DETCA and Tiron had no effect on the relaxations to diethylamine-NONOate or forskolin (P > 0.05, n = 6). Our results demonstrate that endothelium-dependent relaxations mediated by NO. are impaired after the inhibition of Cu/Zn SOD. Relaxations to bradykinin (but not A-23187) were significantly augmented by Tiron. Pharmacological scavenging of O-2. reverses the effect of Cu/Zn SOD inhibition. (+info)
Effect of 3,6-dimethamidodibenzopyriodonium citrate on Ca2+ in rabbit platelet in vitro.
(11/3227)
AIM: To study the effect of 3,6-dimethamidodibenzopyriodonium citrate (I-65) on the cytoplasmic free Ca2+ ([Ca2+]i) concentration in rabbit platelet. METHODS: Measurement of the cytosolic Ca2+ of platelets in vitro by using Quin 2-AM fluorescence technique. RESULTS: In the presence of CaCl2 1 mmol.L-1, I-65 (10, 20, and 30 mumol.L-1) reduced the rise in [Ca2+]i induced by thrombin and calcimycin from 142 +/- 22 nmol.L-1 and 124 +/- 18 nmol.L-1 to 118 +/- 20, 78 +/- 12, 40 +/- 10 nmol.L-1, respectively and 108 +/- 15, 77 +/- 14, 37 +/- 14 nmol.L-1, respectively. In the presence of egtazic acid 2 mmol.L-1, I-65 (10, 20, and 30 mumol.L-1), reduced the Ca2+ release induced by thrombin from 52 +/- 11 nmol.L-1 to 34 +/- 9, 19 +/- 6, and 11 +/- 5 nmol.L-1, respectively. In addition, I-65 (10, 20, and 30 mumol.L-1) also reduced the Ca2+ influx induced by thrombin from 91 +/- 13 nmol.L-1 to 84 +/- 15, 58 +/- 15, and 28 +/- 19 nmol.L-1, respectively. CONCLUSION: I-65 inhibited not only the Ca2+ release, but also the influx of Ca2+ in activation platelet. (+info)
Cyclosporine inhibited calcium-mediated apoptosis of HL-60 cells.
(12/3227)
AIM: To study the effects of cyclosporine (Cyc) on apoptosis of HL-60 cells. METHODS: Apoptotic cells induced by harringtonine (Har), camptothecin (Cam), or calcimycin (Cal), thapsigargin (Tha) were identified with DNA electrophoresis, morphology, and flow cytometry. Relative [Ca2+]i alteration of apoptotic HL-60 cells were determined with flow cytometry. RESULTS: Cal 1 mg.L-1 or Tha 0.5 mg.L-1 induced apoptosis of HL-60 cells. This effect was inhibited by nontoxic concentration of Cyc 1 mg.L-1. Cyc did not inhibit Har- or Cam-induced apoptosis of HL-60 cells. Both Cal and Tha increased intracellular calcium, whereas Har or Cam did not. CONCLUSION: Cyc inhibited apoptosis only induced by calcium increasement in HL-60 cells. The mechanism of apoptosis induced by Cal or Tha was different from that by Har or Cam. (+info)
Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase.
(13/3227)
Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+-calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia. (+info)
Murine cytomegalovirus (MCMV), which causes acute, latent, and persistent infection of the natural host, is used as an animal model of human cytomegalovirus (HCMV) infection. Transcription of MCMV immediate-early (IE) genes is required for expression of the early and late genes and is dependent on host cell transcription factors. Cell-type-specific expression activity of the MCMV IE promoter was analyzed in transgenic mice generated with the major IE (MIE) enhancer/promoter involving nucleotides -1343 to -6 (1338 bp) connected to the reporter gene lacZ. Distinct expression was observed in the brain, kidneys, stomach, and skeletal muscles. Weak expression was observed in a portion of the parenchymal cells of the salivary glands and pancreas, and expression was hardly detected in the lungs, intestine, or immune and hematopoietic organs such as the thymus, spleen, lymph nodes, and bone marrow. The spectrum of organs positive for expression was narrower than that of the HCMV MIE promoter-lacZ transgenic mice reported previously and showed a greater degree of cell-type specificity. Interestingly, astrocyte-specific expression of the transgene was observed in the brain and primary glial cultures from the transgenic mice by combination of beta-galactosidase (beta-Gal) expression and immunostaining for cell markers. However, the transgene was not expressed in neurons, oligodendroglia, microglia, or endothelial cells. Furthermore, the beta-Gal expression in glial cultures was stimulated significantly by MCMV infection or by addition of calcium ionophore. These observations indicated that expression activity of the MCMV IE promoter is strictly cell-type specific, especially astrocyte-specific in the brain. This specific pattern of activity is similar to that of natural HCMV infection in humans. (+info)
Anion efflux from cytotrophoblast cells derived from normal term human placenta is stimulated by hyposmotic challenge and extracellular A23187 but not by membrane-soluble cAMP.
(15/3227)
The regulation of placental anion transport influences fetal accretion and placental homeostasis. We investigated whether efflux of 125I- or 36Cl- from multinucleated cytotrophoblast cells derived from human term placenta is regulated by one of three stimuli: (a) the calcium ionophore A23187, (b) a 'cocktail' of agents designed to raise intracellular levels of cAMP, (c) a hyposmotic solution. After loading with the appropriate isotope for 2 h and thorough washing, cells were exposed to sequential aliquots of buffer applied and removed each minute. Following an equilibration period of 5 min one of the stimuli was applied at room temperature At the end of the experiment the cells were lysed to give a lysate count which was used to express the count obtained from each aliquot as percentage efflux of that possible for that minute. The cAMP 'cocktail' and A23187 were applied for 5 min; the hyposmotic solution was applied for 10 min. The results for 125I- at 7 min showed that the mean efflux in the presence of hyposmotic shock was greater than control (5.7 +/- 1.0% min-1 versus 2.2 +/- 0.1% min-1, respectively; mean +/- S.E.M., n = 4 placentas). Similarly mean efflux at 6 min in the presence of A23187 was also significantly greater than control (6.5 +/- 1.9% min-1 versus 2.6 +/- 1.0% min-1, respectively, n = 3 placentas). The mean efflux in the presence of the cAMP cocktail was not different from control at any time point. The results were qualitatively the same if 36Cl- was used in the place of 125I- and when the experiment was performed with 36Cl- in a HCO3- buffer gassed with CO2. Mean 125I- efflux at 6 min in response to hyposmotic challenge was 33% less (P < 0.01) in the presence of 1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 37% less (P < 0.005) in the presence of 10 microM tamoxifen but no different if the hyposmotic solution was nominally calcium free. We conclude that there are differential effects of second messengers on anion efflux from the differentiated cytotrophoblast cells. (+info)
Calcium-dependent regulation of cytochrome c gene expression in skeletal muscle cells. Identification of a protein kinase c-dependent pathway.
(16/3227)
Mitochondrial biogenesis can occur rapidly in mammalian skeletal muscle subjected to a variety of physiological conditions. However, the intracellular signal(s) involved in regulating this process remain unknown. Using nuclearly encoded cytochrome c, we show that its expression in muscle cells is increased by changes in cytosolic Ca2+ using the ionophore A23187. Treatment of myotubes with A23187 increased cytochrome c mRNA expression up to 1.7-fold. Transfection experiments using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since A23187 increased chloramphenicol acetyltransferase activity by 2.5-fold. This increase was not changed by KN62, an inhibitor of Ca2+/calmodulin-dependent kinases II and IV, and it was not modified by overexpression of protein kinase A and cAMP response element-binding protein, demonstrating that the A23187 effect was not mediated through Ca2+/calmodulin-dependent kinase- or protein kinase A-dependent pathways. However, treatment of myotubes with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c transactivation by 40-50%. Coexpression of the Ca2+-sensitive protein kinase C isoforms alpha and betaII, but not the Ca2+-insensitive delta isoform, exaggerated the A23187-mediated response. The short-term effect of A23187 was mediated in part by mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) since its activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor. These results demonstrate the existence of a Ca2+-sensitive, protein kinase C-dependent pathway involved in cytochrome c expression and implicate Ca2+ as a signal in the up-regulation of nuclear genes encoding mitochondrial proteins. (+info)