Concentrations Of A23187 Research Articles

Involvement of thromboxane A2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation.

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 mM) was inhibited by PAF receptor blocker (WEB 2086, IC50 = 0.65 mM) and calcium channel blockers, diltiazem (IC50 = 13 mM) and verapamil (IC50 = 18 mM). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 mM), a cyclooxygenase inhibitor (indomethacin; IC50 = 0.2 mM) and inhibitor of TLCK, herbimycin A with IC50 value of 5 mM. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 mM. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence

The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.

Activation and penetration in vitro of pig oocytes treated with calcium ionophore.

The present study examined the effects of pre-treatment of pig oocytes with different concentrations (0-50 microM) of calcium ionophore A23187 (CaA) on their activation, development and penetration in vitro. Although untreated oocytes were not activated and did not cleave in culture, high proportions of treated oocytes did so and 20% of oocytes developed to the blastocyst stage when treated with 6.25 microM CaA for 2 min. However, these proportions were reduced in a concentration-dependent manner. When inseminated in vitro with 1 x 10(6) spermatozoa/ml, the penetration rate of oocytes treated with 6.25 microM CaA was similar to that of untreated oocytes. However, fewer oocytes treated with 12.5 and 50 microM CaA were penetrated than untreated oocytes. On the other hand, the proportion of monospermy of oocytes treated with 6.25 microM CaA was higher than the values in oocytes not treated or treated with 50 microM CaA. The time required for zona dissolution of oocytes treated with 6.25 and 12.5 microM CaA was not different from that in untreated oocytes, but oocytes treated with 50 microM CaA required a longer time than untreated oocytes, indicating that zona solubility by protease does not reflect penetrability of oocytes in vitro. When oocytes were inseminated with different concentrations (1-10 x 10(6) cells/ml) of spermatozoa, the highest penetration rate was observed at 1 x 10(6) cells/ml in untreated oocytes and a similar result was obtained in oocytes treated with 6.25 microM CaA. There was no difference in the rate of monospermy in untreated oocytes among different concentrations of spermatozoa, but in treated oocytes, higher proportions of monospermy were observed at 0.5-5 x 10(6) than 10 x 10(6) cells/ml. At 1 x 10(6) cells/ml, the proportion of monospermy was higher in treated than untreated oocytes. These results suggest that pre-treatment of pig oocytes with 6.25 microM CaA, an appropriate concentration, inhibits polyspermic penetration in vitro when insemination occurs with spermatozoa at a concentration of 1 x 10(6) cells/ml.

Downregulation of nitric oxide formation by cytosolic phospholipase A 2-released arachidonic acid

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A 2 (PLA 2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA 2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.

Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation.

Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

High molecular weight DNA fragments are processed by caspase sensitive or caspase independent pathways in cultures of cerebellar granule neurons

Many recent reports on internucleosomal DNA fragments have appeared, however, little is known about the mechanisms of the generation of their upstream high molecular weight (HMW) fragments. Caspases are a family of proteases with important functions in the execution of apoptotic cell death. The caspase-sensitivity of the formation of HMW fragments was therefore investigated using a specific caspase-3 inhibitor (Ac-DEVD-cmk) and a general caspase inhibitor (boc-D-fmk). Apoptosis inducing factor (AIF) can translocate to the nucleus and generate HMW fragments independently of caspase. Cultures of cerebellar granule neurons (CGNs) were therefore exposed to glutamate (100 μM) or deprived of potassium and serum to induce apoptosis, or treated with a high concentration of calcium ionophore A23187 (1 μM) to induce necrosis. Fragmentation of DNA into two classes of HMW fragments (>680 and 50–300 kbp) was observed after treatment with glutamate or A23187. Traces of ∼50-kbp fragments were detectable after the K +/serum-deprivation. The amount of >680-kbp HMW fragments increased (i.e. their further degradation was inhibited) and cell death was reduced in the presence of Ac-DEVD-cmk or boc-D-fmk following glutamate treatment. Only boc-D-fmk treatment resulted in a similar accumulation of >680-kbp HMW fragments and reduced cell death after K +/serum-deprivation. No such changes were observed with caspase inhibitors after A23187 treatment. AIF redistribution was observed following glutamate treatment and K +/serum-deprivation. Thus, even in a simple cell culture of CGNs, HMW fragments are formed by diverse mechanisms: the degradation of DNA may be sensitive to different caspases or be caspase and AIF independent.

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187.

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36 months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60 min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0 microM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3 +/- 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P < 0.001) increased after incubation with A23187. After incubation with 0.1 microM/l A23187 for 45 and 60 min there were 22.4 +/- 3.0% and 31.7 +/- 4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0 microM/l A23187 for 45 and 60 min there were 46.2 +/- 6.5% and 53.8 +/- 5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0 +/- 2.7% and 22.3 +/- 4.2%. There was also a significant (P < 0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.

Effect of Treatment of In Vitro Matured Pig Oocytes with Calcium Ionophore on Monospermic Penetration In Vitro

The present study examined whether treatment of in vitro matured pig oocytes with calcium ionophore (A23187) could prevent polyspermic penetration in vitro. When oocytes cultured for maturation for 33, 36 or 44 h were subsequently treated with 50 µM A23187 in medium with fetal calf serum (FCS) for 1, 2 and 3 h and then cultured for 12 h without spermatozoa, virtually no activation occurred. In the absence of FCS, however, 31-42, 45-49 and 56-64% of oocytes were activated, respectively. When oocytes treated with 50 µM A23187 in medium with FCS for 3 h were inseminated in vitro, the penetration rates (14-57%) were lower (p<0.01) with a higher (p<0.01) incidence (35-67%) of monospermy compared with untreated oocytes (69-80% penetration and 15-17% monospermy). However, sperm penetration was completely blocked in all oocytes treated with A23187 in the absence of FCS. When oocytes matured for 33 h were treated with different concentrations of A23187 for 3 h and inseminated in vitro, the penetration rate did not change but there was an increased incidence (p<0.05) of monospermy at 10-20 µM and 2.5-5 µM A23187 in the presence and absence of FCS, respectively, compared with at 0 µM A23187. With these lower concentrations of A23187, treatment of oocytes for at least 60 and 30 min in the presence and absence of FCS, respectively, was required to increase the incidence of monospermy without reducing penetration rate. These results indicate that a high concentration (50 µM) of A23187 in medium without FCS, but not in medium with FCS, stimulated in vitro matured pig oocytes to induce parthenogenetic activation and a complete block to sperm penetration in vitro. However, treatment of oocytes with lower concentrations of A23187 (10-20 µM and 2.5-5 µM) both in the presence and absence of FCS maintained sperm penetration in vitro and increased the incidence of monospermy. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 2 : 172-178)

Comparative effects of carboxylic ionophores on membrane potential and resistance of NG108-15 cells

Comparative analyses were conducted to determine the effects of Na + (monensin, MON), K + (nigericin, NIG) and Ca 2+ (A23187) selective carboxylic ionophores on differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Alterations in membrane potential (V m), input resistance (Rin) and electrically induced action potential generation were measured using intracellular microelectrode techniques in cells treated with 0.1–30 μ m MON and NIG and 0.1–10 μ m A23187. Responses to the ionophores were similar in that membrane hyperpolarization and unchanged R in predominated with all three compounds. However, significant differences between the ionophores were also detected. MON- and A23187-induced hyperpolarization was generally maintained throughout the 24-min superfusion whereas that produced by NIG diminished with time or was replaced by depolarization. In addition, action potential generation was blocked by NIG, whereas MON had no effect and action potential alterations were evident only with the highest A23187 concentration (10 μ m). This study represents the initial comprehensive analysis of the effects of carboxylic ionophores on membrane electrical characteristics of an intact cell system and forms the basis for subsequent work using NG108-15 cells as a model system to evaluate potential therapeutic treatments against the carboxylic ionophores.

Anisomycin Does Not Activate p38 MAPK in Human Platelets

Human platelets are known to contain three forms of mitogen-activated protein kinases; erk1, erk2, and p38 MAPK. However the role(s) of mitogen-activated protein kinase cascades in platelet function remains to be determined. Evidence has been presented that suggests that these kinases are involved in the cytoskeleton and in the activation of phospholipase A 2; however, other functions seem likely. The object of the present study was to examine the role of the p38 MAPK in platelet function using anisomycin, a reported activator of p38 MAPK, and SB203580, an inhibitor of p38 MAPK. Thrombin and collagen caused the phosphorylation of p38 MAPK and this was inhibited by SB203580. Anisomycin did not cause the aggregation of either intact or saponin-permeabilised platelets. In addition anisomycin failed to produce synergistic aggregation responses with submaximal concentrations of collagen, thrombin, the thromboxane mimetic U46619, or the calcium ionophore A23187. There was no detectable phosphorylation of p38 MAPK in either intact platelets or platelet lysates incubated with anisomycin. Anisomycin also failed to modulate p38 MAPK phosphorylation in response to submaximal concentrations of collagen, thrombin, U46619, or A23187. In contrast anisomycin did cause p38 MAPK phosphorylation in rabbit lung and C3 fibroblasts and in rabbit lung fibroblast lysates. These data demonstrate that anisomycin has no detectable effect on either platelet function or p38 MAPK phosphorylation and, therefore, that anisomycin has proven to be an ineffective tool to define the role that p38 MAPK plays in platelet function.

Influence of cytoplasmic pH on the aggregation and Ca2+ mobilization in rabbit platelets.

The aggregability of rabbit platelets was studied under various cytoplasmic pHs (pHi). Nigericin, a K+/H+ ionophore, which can induce a decrease in pHi, at 2-10 microM in 2 min incubation reduced both platelet aggregation and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) stimulated with thrombin or U46619. The reduced aggregability recovered 10 min after incubation with nigericin in parallel with an increase in pHi. In contrast, when pHi was increased by simultaneous addition of NH4Cl, methylamine or monensin, aggregation in response to a low concentration of thrombin, U46619, arachidonic acid or A23187 was enhanced significantly. The enhancing effect of NH4Cl was lowered by prolonged incubation with NH4Cl, by which the increased pHi was improved concomitantly. Indomethacin, an inhibitor of cyclooxygenase, failed to inhibit the enhancement of aggregation by NH4Cl under stimulation with U46619. In addition, treatment with NH4Cl enhanced an increase in [Ca2+]i in response to U46619 in a concentration-dependent manner, although the treatment by NH4Cl alone did not affect [Ca2+]i. When pHi was artificially altered during the ranges of 6.6-7.4 by treatment with nigericin in K+-rich medium, aggregation by low concentrations of thrombin was dependent on the pHi. These data indicate that pHi is an important factor for platelet activation including intracellular Ca2+ mobilization and aggregation.

Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve.

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.

Evidence for Dimer Participation and Evidence Against Channel Mechanism in A23187-Mediated Monovalent Metal Ion Transport Across Phospholipid Vesicular Membrane

The decay of the pH difference (ΔpH) across soybean phospholipid vesicular membrane by ionophore A23187 (CAL)-mediated H +/M + exchange (M + = Li +, Na +, K +, and Cs +) has been studied in the pH range 6–7.6. The ΔpH in these experiments were created by temperature jump. The observed dependence of ΔpH relaxation rate 1/ τ on the concentration of CAL, pH, and the choice of M + in vesicle solutions lead to the following conclusions. 1) The concentrations of dimers and other oligomers of A23187 in the membrane are small compared to the total concentration of A23187 in the membrane, similar to that in chloroform solutions reported in the literature. 2) In the H + transport cycle leading to ΔpH decay, the A23187-mediated H + translocation across the membrane is a fast step, and the rate-limiting step is the A23187-mediated M + translocation. 3) Even though the monomeric Cal-H is the dominant species translocating H +, Cal-M is not the dominant species translocating M + (even at concentrations higher than [Cal-H]), presumably because its dissociation rate is much higher than its translocation rate. 4) The pH dependence of 1/ τ shows that the dimeric species Cal 2LiLi, Cal 2NaNa, Cal 2KH, and Cal 2CsH are the dominant species translocating M +. The rate constant associated with their translocation has been estimated to be ∼5 × 10 3 s −1. With this magnitude for the rate constants, the dimer dissociation constants of these species in the membrane have been estimated to be ∼4, 1, 0.05, and 0.04 M, respectively. 5) Contrary to the claims made in the literature, the data obtained in the ΔpH decay studies do not favor the channel mechanism for the ion transport in this system. 6) However, they support the hypothesis that the dissociation of the divalent metal ion-A23187 complex is the rate limiting step of A23187-mediated divalent metal ion transport.

Modification of production of reactive oxygen species in mouse peritoneal neutrophils on exposure to low-intensity modulated millimeter wave radiation

The effect of low-intensity modulated electromagnetic radiation of extremely high frequencies (EHF EMR) on the production of reactive oxygen species by mouse peritoneal neutrophils was investigated. The neutrophil activity in synergistic reaction of calcium ionophore A23187 and phorbol ester PMA was estimated by luminol-dependent chemiluminescence technique. The cells were irradiated for 20 min in the far-field zone of the channel radiator in the presence of A23187 and then were activated by PMA. It was shown, that continuous EHF EMR (50 μW cm −2) inhibited quasi-resonantly the synergistic reaction. The maximum effect was about 25% at carrier frequency of 41.95 GHz. The effect had S-like dependence on an absorbed energy flux density, and in a range from 20 to 150 μW cm −2 varied slightly and on the average was about 24%. The modulated radiation with carrier frequency of 41.95 GHz and modulation frequency of 1 Hz activated the synergistic reaction, but at modulation frequencies of 0.1, 16 and 50 Hz inhibited one. At fixed modulation frequency of 1 Hz the nonlinear dependence of the effect on the carrier frequency was found. The synergistic reaction was activated in the frequency range of 41.95–42.05 GHz and was inhibited at the frequencies of 41.8–41.9 GHz. The effect was observed only at high A23187 concentrations used. The obtained results prove the opportunity of control over neutrophil functioning by low-intensity modulated EHF EMR, presumably manipulating by coupled systems of enzyme reactions.

On the expression of cytosolic calcium-independent phospholipase A 2 (88 kDa) in immature and mature myeloid cells and its role in leukotriene synthesis in human granulocytes

The human calcium-independent phospholipase A 2 (iPLA 2; 88 kDa) has recently been cloned (Larsson, P.K.A., Claesson, H.-E. and Kennedy, B.P. (1998) J. Biol. Chem. 272, 207–214). Here we demonstrate the expression of the human iPLA 2 mRNA and its splice variants in blood progenitor cells, immature leukemic cells and mature granulocytes. Chromatographical resolvable iPLA 2 activity was found in the cytosolic fraction of granulocytes and the activity was inhibited by the iPLA 2 inhibitor bromoenol lactone. This drug also inhibited leukotriene synthesis in human granulocytes, induced by low concentration of calcium ionophore A23187 (0.10–0.15 μM) or opsonized zymosan. These results suggest that iPLA 2 is involved in the regulation of the pool of arachidonic acid destined for leukotriene synthesis in human granulocytes.

Parthenogenetic activation of pig oocytes with calcium ionophore and the block to sperm penetration after activation.

Calcium ionophore A23187 can parthenogenetically activate oocytes in many animals. The present study was designed to analyze functionally the mechanism of A23187 activation of pig oocytes matured in vitro. In experiment 1, effects of the concentration of A23187 on intracellular calcium transients, cortical granule (CG) exocytosis, nuclear activation, and zona reaction, which was determined by zona hardening and sperm penetrability, were examined. Cumulus-free oocytes were exposed to 0-100 microM A23187 for 5 min. It was found that the amplitude of the intracellular calcium transients, percentage of CG exocytosis, and percentage of pronuclear formation were increased in a concentration-dependent manner. The time for dissolution of zona pellucida (ZP) was increased in the oocytes treated with 25-100 microM A23187. Penetration of ZP-intact oocytes by spermatozoa was decreased and only 3-4% of oocytes were penetrated by spermatozoa after 50-100 microM A23187 treatment. In experiment 2, oocytes were treated with 100 microM A23187 for 5 min and then cultured for 10 min or 3.5 h before insemination. No difference in penetration rates was observed between the two groups of oocytes (12.0% vs. 12.2%), but the penetration rates were significantly lower than those in controls (85.2% vs. 82.4%). In experiment 3, treatment of oocytes with 100 microM A23187 for 5 min was followed by removal of the ZP from a portion of the oocytes. ZP-intact and ZP-free oocytes were then inseminated for examination of sperm penetration. One of 65 (2%) oocytes with ZP and 48 of 52 (92%) oocytes without ZP were penetrated by spermatozoa. These results indicate that activation of pig oocytes by A23187 is the result of A23187-induced intracellular calcium increase and that A23187-induced cortical reaction can prevent sperm penetration of ZP-intact oocytes, but not ZP-free oocytes.

Possible Mechanism of High Calcium–Induced Relaxation of Rabbit Thoracic Aorta

1. The present study examined the effects of various agents on high calcium–induced relaxation of the rabbit thoracic aorta precontracted by phenylephrine (0.1 μM) or KCl (30 mM). 2. The vascular smooth muscle relaxation caused by high calcium was not changed in the presence of endothelium, glibenclamide (3 μM), TEA (5 mM), verapamil (1 μM), lidocaine (0.1 mM) and vanadate (0.1 mM). 3. In the presence of ouabain (0.1 mM) or potassium-free medium, high calcium–induced relaxation was completely abolished. 4. When rings were precontracted by high concentrations of phenylephrine (1 μM, 10 μM) and KCl (30 mM, 45 mM, 60 mM), calcium-induced relaxation was gradually decreased. 5. A low concentration of calcium ionophore A-23187 (0.1 μM) did not change calcium-induced relaxation, but A-23187 at a high concentration (1 μM) depressed this relaxation. 6. These results suggest that Na-K-ATPase activation could be responsible for high calcium–induced relaxation.

Gradual Reperfusion Reduces Infarct Size and Endothelial Injury but Augments Neutrophil Accumulation

Background. Reperfusion causes injury to the coronary artery endothelium primarily by neutrophil-mediated mechanisms. However, factors other than neutrophils may govern the extent of myocardial necrosis. This study tests the hypothesis that gradual initiation of reflow will reduce reperfusion injury and preserve postischemic endothelial function. Methods. In 16 anesthetized dogs, the left anterior descending artery was ligated for 60 minutes. In one group, reperfusion was initiated abruptly (abrupt, n = 8), whereas in the gradual reperfusion group (ramp, n = 8), flow was slowly initiated during the first 30 minutes of reperfusion. After reperfusion, coronary artery segments were isolated to assess postischemic endothelial function. Results. Infarct size (area of necrosis/area at risk) was significantly reduced in the ramp group (28.2% ± 2.0%) compared with abrupt (41.6% ± 1.4%). Neutrophil accumulation (myeloperoxidase) in the area at risk was significantly greater in the ramp group compared with abrupt (8.0 ± 1.3 versus 3.5 ± 0.8 U/g tissue). In isolated postischemic left anterior descending arterial rings, the concentration of acetylcholine that elicited a response 50% of the maximum possible response was significantly greater in abrupt (−6.88 ± 0.04 log [mol/L]) than ramp (−7.62 ± 0.04 log [mol/L]) and control (−7.68 ± 0.003 log [mol/L]), suggesting endothelial dysfunction. The concentration of A23187 that elicited a response 50% of the maximum possible response was similarly greater in abrupt (−7.24 ± 0.03 log [mol/L]) versus ramp (−7.62 ± 0.03 log [mol/L]) and control (−7.8 ± 0.04 log [mol/L]). Smooth muscle dysfunction (response to sodium nitrite) also occurred in the abrupt rings. Conclusions. Gradual reperfusion of an ischemic area reduces infarct size and preserves endothelial function but paradoxically increases neutrophil accumulation within the area at risk.

Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type 1 and estradiol-17 β-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 β-hydroxysteroid dehydrogenase type 1 (3 β-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 β-hydroxysteroid dehydrogenase type 1 (17 β-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 β-HSD-1 and 17 β-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 β-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 β-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 μM of A23187 the induction of 3 β-HSD-1 and 17 β-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 β-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 β-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 β-HSD-1, 17 β-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 β-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 β-HSD-1 and 17 β-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 β-HSD-1 and 17 β-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

Human endothelial cell activation and mediator release in response to the bacterial exotoxins Escherichia coli hemolysin and staphylococcal alpha-toxin.

Escherichia coli hemolysin (HlyA) and Staphylococcus aureus alpha-toxin are membrane-perturbating bacterial exotoxins that have been implicated as significant virulence factors in human diseases. We investigated the capacity of these toxins to cause cell activation and mediator release in human endothelial cells, compared with the efficacies of thrombin and the Ca2+ ionophore A23187. Concentration ranges tested were 1 to 1000 ng/ml (HlyA), 0.01 to 10 micro/ml (alpha-toxin), 0.01 to 10 U/ml (thrombin), and 0.01 to 10 microM (A23187). All stimuli caused dose-dependent generation of platelet-activating factor, nitric oxide, and prostaglandin I2. HlyA and thrombin effected time- and dose-dependent accumulation of large quantities of inositol phosphates, with maximum effects at 100 ng/ml and 1 U/ml, respectively. Corresponding time course and dose dependency were noted for HlyA-elicited diacylglycerol formation. In contrast, only the highest concentrations of alpha-toxin (10 microg/ml) and A23187 (10 microM) effected some moderate inositol phosphate accumulation, and this was suppressed in the presence of the platelet-activating factor antagonist WEB 2086. Metabolic and secretory responses elicited by alpha-toxin were dependent on the presence of extracellular Ca2+. We conclude that both HlyA and alpha-toxin are potent inductors of inflammatory and vasodilatory mediators in human endothelial cells. HlyA-elicited effects may proceed predominantly via activation of the phosphatidylinositol hydrolysis-related signal transduction pathway, whereas transmembrane Ca2+ flux appears to be the major event underlying the release of mediators in response to alpha-toxin. These toxin properties may contribute to vasoregulatory and inflammatory disturbances encountered in states of severe infection and sepsis.