Swelling-induced O2- generation in guinea-pig neutrophils.

Without the addition of any exogenous stimuli, neutrophils generated O2- and then ceased in a reversible manner that correlated with cellular swelling and contraction. The nature of the possible mechanism responsible for this O2- generation was studied and compared with that observed in the triggering of stimulant-dependent O2- generation (respiratory burst). The swelling-induced O2- generation was inhibited by diphenyliodonium, and was independent of the functional distortion of mitochondrial and/or microsomal electron transport and xanthine oxidase. This suggested that such generation was involved in respiratory-burst oxidase activation; however, this generation was not accompanied by any new phosphorylation of the 47-kDa protein or of tyrosine proteins. Dihydrocytochalasin B potentiated the O2- generation. The cellular swelling produced a priming effect on the triggering of respiratory burst with different stimuli. Cellular contraction, conversely, suppressed the respiratory burst. The structural specificity of the swelling-induced plasma membrane modulation for the O2- generation was suggested by the finding that modulation of plasma membrane structures by various non-ionic detergents per se inhibited O2- generation. Lipophilic and positively-charged agents inhibited the generation and this inhibition was abrogated by negatively-charged, but not by non-ionic agents. Negatively-charged agents potentiated the O2- generation. These results suggest that both the interaction of the plasma membrane with the cytoskeleton and an increase in net negative charges at the plasma membrane play important role in evoking O2- generation; this is discussed and compared with the signal transduction reported previously for respiratory burst.

Okadaic acid increased annexin I and induced differentiation of human promyelocytic leukemia cells.

The differentiation of a cell line of human promyelocytic leukemia, HL-60 cells, triggered by 12-O-tetradecanoyl 13-phorbol acetate (TPA), depends on the phosphorylation of some proteins, such as 17, 27, and 34 kDa proteins, by protein kinase C. For elucidation of the mechanism of ligand-induced differentiation of HL-60 cells, the effects of okadaic acid (OA), a phosphatase inhibitor, on cell differentiation and protein phosphorylation were studied. After treatment with OA, HL-60 cells differentiated into macrophage-like cells; within 16 h, 70% or more of the treated cells adhered to plastic dishes. The adherent cells did not undergo mitosis but began activities such as phagocytosis. OA increased the phosphorylation of 17, 23, 27, and 34 kDa proteins, as did TPA. The amount of annexin I (39 kDa protein) in HL-60 cells caused to differentiate with OA was 7.5-fold that without such treatment. Kinetic analysis showed that increased transcription of annexin I mRNA caused the increase in annexin I in the differentiated cells. Thus, OA and TPA increased cellular levels of annexin I and caused the differentiation of HL-60 cells into macrophage-like cells.

Dynamic aspects of ovarian superoxide dismutase isozymes during the ovulatory process in the rat.

To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rats. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.

Expression of the cDNA encoding lipocortin-like 39 kDa protein of guinea pig neutrophils in yeast. Purification and biological characterization.

The cDNA encoding lipocortin-like 39 kDa protein in guinea pig neutrophils was cloned into a yeast expression vector and the constructed plasmid was introduced into a yeast. The gene was expressed in an eukaryotic cell, yeast Saccharomyces cerevisiae and the recombinant protein was purified and characterized. The purified protein was identical with the native one with respect to the antigenicity and several biochemical properties, such as inhibitory action against phospholipase A2, Ca2+-dependent binding to acidic-phospholipids and F-actin and availability as a substrate for tyrosine kinase (EGF receptor/kinase) and protein kinase C.

Inhibition of 12-O-tetradecanoyl phorbol-13-acetate promoted tumorigenesis by cepharanthine, a biscoclaurine alkaloid, in relation to the inhibitory effect on protein kinase C.

In two-stage mouse skin carcinogenesis initiated by 7,12-dimethylbenz[alpha]anthracene (DMBA), cepharanthine inhibited the tumor promoting activity of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Since Ca2(+)-phospholipid-dependent protein kinase (PKC) was shown to be an intracellular target of TPA, effects of cepharanthine on the activity of this enzyme were investigated Cepharanthine also inhibited the phosphorylation of H1 histone by PKC in a concentration dependent manner. While cepharanthine inhibited the association of H1 histone with phospholipid vesicles, autophosphorylation of PKC was not inhibited by this drug. Cepharanthine also inhibited TPA-stimulated phosphorylation of some cytoplasmic proteins of mouse skin epidermis. These results indicated the possibility that anti-tumor promoting action of cepharanthine was the result of inhibition of PKC dependent cytoplasmic protein phosphorylation through the reduction of the interaction of these proteins with the plasma membrane.

Inhibition of active oxygen generation in guinea-pig neutrophils by biscoclaurine alkaloids.

Effect of biscoclaurine alkaloids, such as cepharanthine, on active oxygen production of neutrophils was investigated. Cepharanthine inhibited both superoxide generation and luminol-dependent chemiluminescence (CL) induced by either formylmethionyl-leucyl-phenylalanine, opsonized zymosan, arachidonic acid or by phorbol myristate acetate. Ca2(+)- and phospholipid-dependent protein kinase (PKC) activity and the phosphorylation of cytoplasmic protein including 47 kDa proteins of neutrophils were also inhibited by cepharanthine; dose dependent inhibition of CL was quite similar to that of PKC. Among various biscoclaurines tested, the inhibitory effect of cepharanthine, tetrandrine and isotetrandrine was strong, but that of berbamine and cycreanine was weak; the inhibitory action of the former on lipid peroxidation and platelet aggregation were also stronger than those of the latter. These and other observations indicated that these alkaloids inhibited the active oxygen generation by way of stabilizing plasma membrane and inhibiting PKC and NADPH oxidase activation.

Photoactivated inhibition of superoxide generation and protein kinase C activity in neutrophils by blepharismin, a protozoan photodynamically active pigment.

Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma. This pigment inhibited the generation of superoxide anion (O2-.) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570 nm but not above 610 nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration-dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.

Activation of protein kinase C by myristate and its requirements of Ca2+ and phospholipid.

Myristate (C14:0) was found to significantly activate partially purified rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC). The Ka value, the concentration needed for half maximum activation, for C14:0 in the presence of 1 microM Ca2+ and 20 microM phosphatidylserine (PS) was 20 microM. This activation required Ca2+ and acidic phospholipid and was associated with a decreased Ka for Ca2+ of the enzyme to 10 microM in an analogous fashion as dioleoylglycerol (DO) or phorbol myristate acetate (PMA). The phospholipid requirement for the activation was concentration dependent and was inhibited by 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H-7), a inhibitor of this enzyme. The concentration of H-7 required for half inhibition of the enzyme was about 15 microM and maximum inhibition was about 75%. The concentration profile of cytoplasmic proteins phosphorylated by C14:0-activated PKC was similar to that by PMA-activated PKC. The 47 kDa protein of guinea pig neutrophil was also phosphorylated by the C14:0-activated PKC. It is further discussed whether PKC can function as signal transduction for stimulus-mediated generation of superoxide in neutrophils.

Local anesthetics enhance nitric oxide production by human peripheral neutrophils.

Various neutrophil functions are suppressed by local anesthetics. We studied the effect of local anesthetics on nitric oxide (NO) generation in human peripheral neutrophils. Lidocaine and other local anesthetics stimulated NO generation in resting neutrophils. Canavanine, an NO synthase inhibitor, inhibited NO generation. Lidocaine and the other local anesthetics enhanced formylmethionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA)-induced NO generation. These findings suggest that NO mediates various pharmacological effects of the local anesthetics on the host defense mechanism and the control of blood pressure.

Increase in cell responses and protein phosphorylation of neutrophils induced by synthetic diacylglycerols.

Diacylglycerols (DG) induced various stimulation-coupled responses of guinea pig peritoneal neutrophils (GPPMN), such as superoxide (O2-.) generation, luminol chemiluminescence response (LCL) and membrane depolarization. These activities induced by L-alpha-1,2-dioctanoyl glycerol (diC8) are quite similar to those induced by phorbol 12-myristate 13-acetate (PMA). The effects of diC8 were much stronger than those of L-alpha-1-oleoyl-2-acetylglycerol (OAG) and no effect was produced by L-alpha-1,2-dioleoyl glycerol (DO). Concentrations of diC8 and OAG for maximal O2-. generation were 1 and 10 microM, respectively. However, rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC) was activated by all DGs used, and the concentrations of OAG, DO and diC8 for half maximum stimulation were 0.2, 1.0 and 2.0 microM, respectively. Discrepancies between the concentrations of DGs required for O2-. release and PKC activation might be due to differences in their affinity for and permeability in the membrane. The generation of active oxygen and the PKC activity were both sensitive to PKC inhibitors, such as 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7) and staurosporine. ID50 of H-7 and staurosporine for the inhibition of diC8-induced O2-. release were 150 nM and 10 nM, respectively. By contrast, staurosporine does not inhibit diC8 induced membrane depolarization. Phosphorylation of cytoplasmic proteins, such as the 47 kDa protein, was increased by DGs and this phosphorylation was also inhibited by H-7 or staurosporine. The capacities of stimulating the rates of 47 kDa protein phosphorylation were in the order diC8 > OAG > DO. These results suggest the involvement of protein phosphorylation in DG-induced O2-. generation. However, a part of the O2-. generation induced by high concentration of DG might have occurred via a PKC-independent pathway.

Metabolic response of polymorphonuclear leukocyte to arachidonic and linoleic acids.

Various stimuli act on polymorphonuclear leukocytes (PMN), activating membrane-bound phospholipase A2 and C, and diglyceride lipase and then liberating unsaturated fatty acids (USFAs). These liberated USFAs are immediately metabolized through various metabolic pathways such as cyclooxygenase, lipoxygenase, phosphatidylinositol metabolism etc. It is possible that the metabolic intermediates of these pathways reveal various physiological actions. This work was undertaken to clarify whether stimuli on PMN depend on these USFAs themselves or on their oxidation products. The following results were obtained: 1. USFAs such as arachidonate and linoleate stimulate PMN, accelerating superoxide (O2) generation, depolarization of membrane potential and increase in [Ca2+]i. 2. Oxidation products of USFAs have no stimulative effect on PMN. The decrease in the stimulative effect of these USFAs following their oxidation is proportional to the quantitative decrease in non-oxidized linoleate. 3. USFAs accelerate membrane permeability of Ca2+, and their oxidation products enhance non-specific membrane permeability in proportion to the formation of monohydroxy compound. These results suggest that stimulative effects of USFAs on PMN do not depend on their oxidation products but on unoxidized fatty acids. Furthermore, among the oxidation products of the USFAs, monohydroxy compound acts as a strong perturber of membrane and accelerates membrane permeability.

Anomalous flexor pollicis longus muscle.

This paper presents a case in which an anomalous tendon from the flexor pollicis longus to the flexor indicis profundus caused limitation of independent flexion of the thumb. The anomaly was accompanied by chronic tenosynovitis.

Expression of mRNAs of the aquaporin family in mouse oocytes and embryos.

The molecular basis of water and cryoprotectant permeability in mammalian oocytes and embryos is poorly understood. Therefore, we investigated the expression of mRNAs of water channel proteins (aquaporins) in mouse oocytes and embryos by RT-PCR. The total RNA of mouse oocytes at metaphase II and embryos at the 4-cell, morula, and blastocyst stages was isolated, reverse-transcribed, and subjected to nested PCR amplification. Aquaporins were expressed in both oocytes and embryos, but the types were different among the developmental stages: aquaporins 3 and 7 were expressed in oocytes and embryos at all stages examined, but aquaporins 8 and 9 were expressed only in blastocysts. On the other hand, aquaporins 1, 2, 4, 5, and 6 were not detected in any of the stages examined. The present study shows for the first time that aquaporins are expressed in mammalian oocytes and embryos. These aquaporins may play a role in water transport and conceivably also in cryoprotectant transport across the plasma membrane in these cells.

Restoration of resistance to osmotic swelling of vitrified mouse embryos by short-term culture.

In cryopreservation of mammalian embryos, embryos can be injured by osmotic swelling during removal of the cryoprotectant after warming. We have shown that vitrified embryos are more sensitive to osmotic swelling than fresh cells but that sensitivity is reduced or abolished if vitrified cells are cultured for a short period before subjecting them to hypotonic stress. In the present study, we examined the mechanism by which vitrified two-cell mouse embryos regain their resistance to osmotic swelling by culturing the embryos in the presence of various inhibitors before hypotonic treatment. New synthesis of RNA and proteins during culture was not required for regaining resistance to osmotic swelling because actinomycin D and cycloheximide failed to inhibit restoration. Inhibitors of polymerization of microfilaments and microtubules (cytochalasin B and demecolcine, respectively) also did not affect restoration of resistance to osmotic swelling, suggesting that rearrangement or repolymerization of cytoskeletal components is not involved in this process. On the other hand, brefeldin A and concanamycin A, which inhibit intracellular vesicular transport, strongly suppressed restoration of resistance. These results suggest that the intracellular vesicular transport system plays a crucial role in restoration of resistance of vitrified embryos to osmotic swelling during short-term culture.

Activation of protein kinase C by fatty acids and its dependency on Ca2+ and phospholipid.

Ca2+- and phospholipid-dependent protein kinase (PKC) was activated by arachidonic and myristic acids. This activation by both fatty acids required the calcium ion. Acidic phospholipid was also required for the activation by myristic acid, while that by arachidonic acid was inhibited by phospholipid.

Differentiation of HL-60 cells by phorbol ester is correlated with up-regulation of protein kinase C-alpha.

To clarify the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of HL-60 cells, we investigated the correlation between the effects of protein kinase C (PKC) inhibitors on the induction of markers of TPA-induced differentiation and those on suggested critical steps of the differentiation. H-7, sphingosine, and trifluoroperazine significantly suppressed TPA-induced cell adhesion but their effects on the induction of acid phosphatase and nonspecific esterase differed among the inhibitors. The three inhibitors failed to affect on TPA-induced annexin I expression. In contrast, staurosporine markedly suppressed the induction of all these markers. The effects of the inhibitors on some suggested critical steps of the differentiation, a rapid phosphorylation of specific proteins, a rapid membrane association of PKC, and down-regulation of PKC at 18 h after addition of TPA, were not correlated with those on the differentiation marker induction. Only the effect of the inhibitors on up-regulation of PKC-alpha was closely correlated with TPA-induced annexin I expression; staurosporine inhibited up-regulation of PKC-alpha but other inhibitors did not similarly affect the induction of annexin I expression. These results suggest that PKC-alpha is intimately related to macrophage-like differentiation of HL-60 cells by TPA.

Reversible priming and protein-tyrosyl phosphorylation in human peripheral neutrophils under hypotonic conditions.

Hypotonic shock enhanced both formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2-.) generation and tyrosyl phosphorylation of cellular proteins including 120-, 115-, 83-, 63-, and 54-kDa proteins of human peripheral neutrophils. The time course of the enhancement correlated with that of tyrosyl phosphorylation of the 115-kDa protein. The "primed state" was reversed to the nonprimed resting state by changing the conditions from hypotonic to isotonic, with a concomitant decrease in tyrosyl phosphorylation. Genistein inhibited the increase in both O2-. generation and tyrosyl phosphorylation of the 120-, 115-, 63-, and 54-kDa proteins. These results suggest the involvement of tyrosyl phosphorylation of a cellular protein(s) in hypotonic shock-induced priming of neutrophils.

Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.

Cryopreservation of zona-hatched mouse blastocysts.

Experiments were conducted to determine the conditions for successful and efficient cryopreservation of hatched mouse blastocysts, using simple vitrification procedures. Hatched blastocysts were obtained by culture of morulae in vitro. Vitrification solutions used were EFS40 and GFS40, which were 40% (v/v) ethylene glycol and 40% (v/v) glycerol, respectively, diluted in PB1 medium containing 30% Ficoll (w/v) and 0.5 mol sucrose l-1. In the one-step method, embryos were directly exposed to the vitrification solutions at 25 degrees C for 0.5 or 2 min; in the two-step method, embryos were equilibrated with a dilute (10-20%, v/v) ethylene glycol or glycerol solution for 5-10 min, before a 0.5 min exposure to EFS40 or GFS40, respectively. They were then vitrified in liquid nitrogen. When the embryos were vitrified in EFS40, the post-warming survival rates, assessed by the re-expansion of the blastocoel during 16 h of culture, were higher in embryos that had hatched from the zona earlier (120-132 h after hCG) than in those hatched later (142-150 h after hCG); however, the highest survival rate was only 65%, which was obtained by a one-step method. When embryos were vitrified in GFS40, a high survival rate (89-94%) was obtained especially by the two-step methods. Vitrified blastocysts developed into live young just as well as did fresh blastocysts; survival was highest after transfer to recipients on day 3 or day 4 of pseudopregnancy. These findings show that hatched mouse blastocysts can be successfully cryopreserved by a simple vitrification method, and that a glycerol-based vitrification solution is more suitable than the corresponding ethylene glycol-based solution for the vitrification, probably because slower permeation of glycerol avoids toxic injury.

Fracture damage of embryos and its prevention during vitrification and warming.

The frequency of fracture damage in mouse blastocysts was examined by repeated cycles of vitrification and warming. Mouse blastocysts suspended in a solution of ethylene glycol, Ficoll, and sucrose in a straw were plunged into liquid nitrogen either directly (rapidly) or after holding them in liquid nitrogen vapor for 3 min or more (moderately). Vitrified samples were warmed by plunging them into 25 degrees C water either immediately (rapidly) or after holding in air for 5-30 s (moderately). Warmed straws were recooled and rewarmed up to 9 times, to exaggerate the effect of cooling and warming. When embryos were cooled and warmed rapidly once, the incidence of the zona damage was only 1.2%, and 91% of recovered embryos reexpanded in culture. However, with repeated rapid cooling and warming, the incidence of zona damage increased, reaching 75% after 10 vitrifications; survival also dropped. When embryos were subjected to 10 cycles of moderate cooling and moderate warming with 15 or 30 s of suspension in air, 100% of the embryos had intact zonae. On the other hand, with moderate cooling followed by rapid warming or with rapid cooling followed by moderate warming, 41 and 16% of embryos had damaged zona, respectively, after 10 vitrifications. Therefore, fracture damage occurs during both cooling and warming, but it can be prevented completely by employing somewhat slower rates of cooling and warming. Furthermore, a high survival rate (88%) after 10 cycles of moderate cooling and moderate warming with 15 s of suspension in air indicates that vitrification, melting, or temperature fluctuation per se do not affect embryonic survival.