Search PubMed⌕ Search

Biomedical subjects

P Blackmore

Publications and source records attributed to P Blackmore.

6 recordsLinked to original sources
PubMed2001

2-Aminoethoxydiphenyl borate directly inhibits store-operated calcium entry channels in human platelets.

In this study, we examined 2-aminoethoxydiphenyl borate (2APB) as an inhibitor of Ca(2+) influx in human platelets. 2APB was found to inhibit thrombin-mediated intracellular Ca(2+) mobilization rapidly in platelets incubated in the absence of extracellular Ca(2+). This result supports an intracellular action of 2APB on inositol 1,4,5-trisphosphate (IP(3))-receptor Ca(2+) channels. 2APB was without effect on the ability of thapsigargin to mobilize intracellular Ca(2+). This result suggests that the efflux of Ca(2+) from the endoplasmic reticulum mediated by thapsigargin is not via IP(3) Ca(2+) channels. However, 2APB was able to prevent the entry of Ca(2+) and Sr(2+) through thapsigargin-activated, store-operated Ca(2+) channels (SOCC). This result supports a direct inhibitory effect of 2APB on SOCC. 2APB was also able to block the entry of Sr(2+), Ba(2+), and Mn(2+) entry into unstimulated platelets, which suggests that 2APB was inhibiting the Ca(2+) influx channels directly. The capacity of 2APB to prevent Ca(2+) influx and Sr(2+) influx was rapid because it occurred immediately upon addition to the platelets. The inhibition of Ca(2+) and Sr(2+) influx by 2APB was similar to that seen with the cell-impermeable nonselective Ca(2+)-channel blocker La(3+) or the Ca(2+) chelator EGTA. Diphenylboronic anhydride and 2,2-diphenyltetrahydrofuran, two compounds that are structurally similar to 2APB, also inhibited Ca(2+) influx. It was concluded that 2APB was a rapid and effective direct inhibitor of SOCC in human platelets; as such, it cannot be used to support the involvement of IP(3) receptors in the activation of SOCC.

Barium↗
PubMed1998

Increase of intracellular calcium is not a cause of pentoxifylline-induced hyperactivated motility or acrosome reaction in human sperm.

OBJECTIVE: To investigate the effects of the phosphodiesterase inhibitor pentoxifylline on hyperactivated motility and acrosome reaction in human sperm and to determine whether its stimulatory effects occur via increased intracellular calcium levels. DESIGN: Prospective study. SETTING: Academic tertiary care facility. PARTICIPANT(S): Healthy male donors. INTERVENTION(S): The effects of pentoxifylline on hyperactivated motility, acrosome reaction, and intracellular calcium were studied and compared with the effects of progesterone. Thapsigargin, a known mobilizer of intracellular calcium, also was used as positive control. MAIN OUTCOME MEASURE(S): Hyperactivated motility was assessed by computer-assisted sperm motion analysis using the HTM-IVOS, acrosome reaction was evaluated with the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum agglutinin, and intracellular calcium was determined by fura-2 using spectrofluorometry. RESULT(S): Pentoxifylline significantly increased both hyperactivated motility and acrosome reaction. Enhancement of hyperactivated motility by pentoxifylline in the capacitation medium persisted for up to 5 hours after pentoxifylline was washed from the medium. It also enhanced the percentage of acrosome-reacted spermatozoa after 4 hours of incubation. These effects occurred in the presence of a marginally significant decrease in intracellular calcium. CONCLUSION(S): Pentoxifylline stimulates hyperactivated motility and acrosome reaction in spermatozoa from fertile men. Its stimulatory effects occur through mechanism(s) other than increase in intracellular calcium.

Acrosome↗
PubMed1995

Effects of hydrogen peroxide on human spermatozoa.

PURPOSE: Reactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (H2O2) may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H2O2 on functions crucial to fertilization in human spermatozoa. METHODS: In these prospective studies, we examined the dose- and time-dependent effects of H2O2 on sperm membrane-mediated events (binding to the zona pellucida and changes in intracellular calcium concentration [Ca2+]i, motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash/swim-up. [Ca2+]i was measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosome reacted sperm was detected by FITC-Pisum sativum lectin and indirect immunofluorescence. RESULTS: In the HZA, H2O2 did not influence sperm-zona pellucida binding at low concentrations (0.05 mM and 0.1 mM), but significantly reduced binding at 0.2 mM (P < 0.004 vs controls). H2O2 significantly decreased HA in a dose-dependent manner (P < 0.0001) and had a significant effect (P < 0.01) on acrosome reaction (stimulatory effect at 0.01 mM). H2O2 did not affect basal [Ca2+]i; however, H2O2 (0.1 mM through 10 mM) decreased the initial phase of progesterone-induced (P4: 1 microM) enhancement of [Ca2+]i in a dose- and time-dependent fashion. Preincubation of sperm with catalase (20 micrograms/ml) potentiated the P4-induced increase of [Ca2+]i. H2O2 did not significantly modify [Ca2+]i increase in response to inomycin (10 microM). CONCLUSIONS: These experiments show that H2O2 directly affects sperm functions crucial to fertilization in a dose- and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intracellular homeostasis by the oxidative process.

Acrosome↗
PubMed1995

Investigation of some biochemical and functional effects of cryopreservation of human spermatozoa using an automated freezing-quick-thawing method.

The objective of the present studies was to assess the functional integrity of the sperm plasma membrane and metabolic and motility characteristics of the recovered motile fraction of human spermatozoa subjected to an automated freezing/quick-thawing method. Sperm membrane features examined included progesterone-induced changes in intracellular levels of calcium ([Ca2+]i), as measured by the fluorescent fura-2 indicator, and the tight binding of spermatozoa to homologous zonae pellucidae as assessed by the hemizona assay (HZA). Basal [Ca2+]i intracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels determined using chemiluminescence with luciferin-luciferase, and motility parameters determined using a computer-aided semen analyser (CASA) were studied concomitantly as an expression of metabolic/functional status. Ejaculates from fertile men (donors) were evaluated after swim-up separation of the motile fraction in both fresh and cryopreserved-thawed samples, and fractions of each ejaculate (fresh and frozen-thawed) were subjected to parallel measurements of the same parameters at the same time frame. Basal and progesterone-induced increase in [Ca2+]i, and ATP levels (up to 24 h) were similar in fresh and frozen-thawed samples. HZA results showed a modest (26%) although significant (p = 0.008) decrease in binding in frozen-thawed samples. The ratios of ATP/ADP in fresh and frozen-thawed samples were also found to be similar. Although post-thaw sperm motility was significantly lower than that of the fresh samples, comparison of the results indicated that the method was capable of preserving > 65% of motile spermatozoa in almost all of the samples cryopreserved. Additionally, the swim-up rescued a motile fraction in the frozen-thawed samples that was not significantly impaired with regard to motility, mean linear velocity or linearity as compared to the fresh fractions in the first 4 h. Our results show that this automated freezing-quick-thawing method results in a small reduction in sperm-zona binding capacity, and that the time-dependent decline in motility parameters observed for both fresh and cryopreserved-thawed samples cannot be related to ATP deficiency under the conditions of our experiments. These in-vitro results are coincident with the maintenance of fertilizing capacity for donor spermatozoa in the in-vitro fertilization (IVF) setting.

Adenosine Diphosphate↗
PubMed1994

Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia.

OBJECTIVE: To evaluate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms. DESIGN: Prospective, controlled study. SETTING: Academic tertiary assisted reproduction center. PATIENTS: Patients (n = 14) were allocated in the study after semen evaluation showed teratozoospermia (< 14% normal sperm forms) as diagnosed by strict criteria. INTERVENTIONS: After swim-up separation of the motile fraction, acrosome reactions were evaluated using Pisum sativum agglutinin (both spontaneously and exogenously induced with P and the calcium ionophore A23187, both at 10 microM); the intracellular-free [Ca2+]i was assessed by the fluorescent fura-2 indicator (basal and after P). RESULTS: Patients did not show the typical P-induced wave of [Ca2+]i that was observed in controls but rather a blunted response, no response at all, or abnormal basal [Ca2+]i levels. The percent of basal acrosome reaction was significantly lower for patients versus controls postswim-up, and at 1 hour and 3 hours. Furthermore, there was a significant difference in the response of acrosome reaction to P both at 1 hour and 3 hours, with patients showing almost no response at all. However, patients' acrosome reaction response to the calcium ionophore was similar to those of fertile men. CONCLUSION: Infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of spontaneous acrosome reaction and a diminished P-stimulated acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Parallel abnormalities of [Ca2+]i were observed in patients, suggesting that these sperm populations may have a defective nongenomic P sperm receptor and/or abnormalities of other membrane transduction systems.

Acrosome↗