Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

Interactions between a decapacitation factor and mouse spermatozoa appear to involve fucose residues and a GPI-anchored receptor

Epididymal mouse spermatozoa have a surface-associated decapacitation factor (DF) that can be removed precociously by centrifugation, resulting in acceleration of capacitation and increased fertilizing ability. Addition of exogenous DF to capacitated suspensions inhibits fertilizing ability and reverses capacitation in acrosome-intact cells. DF appears to regulate a Ca²⁺-ATPase, located primarily in the postacrosomal region. The present investigations of DF↮spermatozoon interaction indicate that DF can be removed from uncapacitated cells by treatment with phosphatidylinositol-specific phospholipase C (PIC), suggesting the involvement of a glycosylphosphatidylinositol (GPI) moiety. However, exogenous DF cannot reassociate with PIC-treated spermatozoa, suggesting that DF may bind to spermatozoa via a GPI-anchored receptor. DF binding appears to involve fucose residues, since depletion of endogenous DF followed by brief exposure to fucose (0.1–10 mM) prevented DF reassociation with cells. Furthermore, 5 mM fucose could displace DF from uncapacitated cells, accelerating capacitation and resulting in a higher proportion of fertilized oocytes, with increased polyspermy, than obtained with untreated controls. FITC-labelled fucosylated BSA bound specifically to the postacrosomal region, binding being inhibited by both excess fucose and crude DF. UEA I, a lectin with specificity for fucose residues, bound to the postacrosomal region of cells preincubated in fucose but not crude DF, and blocked DF binding to DF-depleted cells. These results are consistent with the DF binding, via fucose residues, to a GPI-anchored receptor. Fucose binding sites are in the same region where Ca²⁺-ATPase, the enzyme regulated by DF, has been localized; these results support the hypothesis that DF modulates capacitation by regulating enzyme activity and hence the intracellular Ca²⁺ concentration. Mol. Reprod. Dev. 51:193–202, 1998. © 1998 Wiley-Liss, Inc.     

Ca2+-Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca²⁺ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: “F,” characteristic of uncapacitated, acrosome-intact cells; “B,” characteristic of capacitated, acrosome-intact, cells; “AR,” characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently ～ 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells. In the presence of either quercetin, a Ca-ATPase inhibitor used at 50–200 μM, or W-7, a calmodulin antagonist used at 5–125 μM, capacitation per se was accelerated, as evidenced by significant decreases in F and significant increases in B pattern cells; only the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 μM W-7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na⁺ ionophore monensin at 500 μM significantly accelerated both capacitation and acrosomal exocytosis. The addition of the dihydropyridine calcium channel blocker nifedipine at 10 nM, just prior to monensin, did not inhibit capacitation (F to B transition) but blocked acrosomal exocytosis (B to AR transition). We suggest that Ca²⁺ is required for functional changes in bull sperm, with a Ca²⁺-ATPase modulating intracellular Ca²⁺ during capacitation and calcium channels controlling the Ca²⁺ influx required for acrosomal exocytosis. © 1995 Wiley-Liss, Inc.     

Mitochondrial movement during the ascidian sperm reaction

The ascidian sperm reaction, Which involves swelling, migration, and loss of the single large mitochondrion, can be triggered in vitro by raising the seawater pH to 9.3 or lowering Na⁺ to 20 mM, but only if the sperm are allowed to attach to a suitable Substate. Mitochondrial translocation does not usually occur in the absence of sperm attachment. Extracellular Ca²⁺ is necessary for triggering the reaction with low Na⁺ but not high pH; however, the intrecellular Ca²⁺ blocker, TMB-8, inhibits high pH-induced mitochondrial movement in the absence of extracellular Ca²⁺. After swelling, the mitochondrion fluoresces in the presence of chlortetracycline, suggesting that Ca²⁺ becomes membranebound after activation. Elevated cAMP and theophylline both inhibit mitochondrial move ment but not sperm motility. The antiactin drug cytochalasin B(10μM) and the calmodulinblocking drugs TFP (1 μM) and W-13 (10 μM) block mitochondrial movement, suggesting roles for actin and calmodulin in mitochondrial movement. A model is proposed relating intracellular alkalinization, Ca²⁺ influx, actin, myosin, and calmodulin in mitochondrial translocation.     

Polyamine transport regulation by calcium and calmodulin: Role of Ca2+-ATPase

The study was conducted on human leukemia (K 562) cells to characterize the mechanisms implicated in the regulation of the polyamine spermicine (Spd) transport process. The antagonists of calmodulin, trifluoperazine (TFP), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthelenesulfonamide), or mellitin inhiblted significantly polyamine Spd uptake in these cells. The translocation of calmodulin towards plasma membrane and a concomitant decrease in its contents in cytosol were directly correlated with the time course increases similar to that of Spd uptake, indicating that calmodulin is recruited towards plasma membrane during the Spd transport process. Diminution of free intracellular calcium, (Ca²⁺)i, by preincubating the cells in BAPTA (bis[2-amino-5-methylphenoxyl]-ethane-N,N′,N′,-tetraacetate) buffer inhibited Spd transport significantly. Addition of lanthanum (LAN), a molecule known to inhibit Ca² efflux via Ca²⁺-ATPase, curtailed Spd uptake by these cells. LAN inhibited Vmax, but not the Km, of Spd uptake, indicating that the former does not directly interact with the polyamine transporter; rather it regulates the transport process, probably via its action on Ca²⁺-ATPase. Calmodulin-stimulated uptake of ⁴⁵Ca²⁺ by inside-out vesicles of K 562 cells, a measure of Ca²⁺-ATPase activity. Furthermore, addition of LAN inhibited both basal and calmodulin-stimulated activity of Ca²⁺-ATPase. Thapsigargin (THAP), a molecule known to elevate (Ca²⁺) i due to its action on the endoplasmic reticulum, increased Spd transport whereas addition of LAN inhibited THAP-stimulated Spd transport activity. THAP increased free (Ca²⁺)i in these cells, and a pre-addition of LAN to these cells curtailed the THAP-stimulated increases of (Ca²⁺)i concentrations. Addition of Spd brought about elevations in (Ca²⁺)i contents. Caffeine also increased (Ca²⁺)i in these cells; however, it failed to stimulate significantly the Spd uptake process, indicating that (Ca²⁺)i which is involved in the regulation of polyamine transport pathways does not belong to the calcium-induced calcium-release (CICR) pool. Replacement of Ca²⁺ from the incubation medium (i.e., 0% Ca²⁺) resulted in higher uptake activity as compared to that in 100% Ca²⁺ medium, demonstrating that in 100% Ca²⁺ medium the calcium efflux process is quickly compensated by calcium refilling/influx from the extracellular medium, while in 0% Ca²⁺ medium there is perpetual efflux of (Ca²⁺)i which contributes to higher Spd uptake process. The results of this study suggest that an increase in free (Ca²⁺)i and its release from the cells via Ca²⁺ ATPase, and concomitant activation of calmodulin, which controls Ca²⁺-pump activity, are involved in the regulation of the Spd uptake process in human leukemia cells. © 1993 Wiley-Liss, Inc.     

Calmodulin blocker inhibits Ca++-ATPase activity in secretory ameloblast of rat incisor

Summary The effects of the calmodulin blocker, trifluoperazine (TEP), on membrane-bound Ca⁺⁺ -ATPase, Na⁺ -K⁺ -ATPase (EC 3.6.1.3.) and the ultrastructure of the enamel organ were investigated in the lower incisors of normal and TFP-injected rats. The rats, of about 100 g body weight, were given either 0.2 ml physiological saline or 100 g TFP dissolved in 0.2 ml physiological saline through a jugular vein and fixed by transcardiac perfusion with a formaldehyde-glutaraldehyde mixture at 1 and 2 h after TFP administration. Non-decalcified sections of the enamel organ less than 50 m in thickness, prepared from dissected lower incisors, were processed for the ultracytochemical demonstration of Ca⁺⁺-ATPase and Na⁺-K⁺ -ATPase by the one-step lead method at alkaline pH. In control saline-injected animals the most intense enzymatic reaction of Ca⁺⁺-ATPase was demonstrated along the plasma membranes of the entire cell surfaces of secretory ameloblasts. Moderate enzymatic reaction was also observed in the plasma membranes of the cells of stratum intermedium and papillary layer. Reaction precipitates of Na⁺-K⁺-ATPase activity were localized clearly along the plasma membranes of only the cells of stratum intermedium and papillary layer. The most drastic effect of TFP was a marked disappearance of enzymatic reaction of Ca⁺⁺-ATPase from the plasma membranes of secretory ameloblasts, except for a weak persistent reaction in the basolateral cell surfaces of the infranuclear region facing the stratum intermedium. The cells of stratum intermedium and papillary layer, however, continued to react for Ca⁺⁺-ATPase even after TFP treatment. Similarly, Na⁺-K⁺-ATPase activity in these cells was not inhibited by TFP administration. Ultrastructural examination of secretory ameloblasts revealed that administration of TFP caused no considerable cytological changes and did not act as a cytotoxic agent. These results suggest that secretory ameloblasts may have an active Ca⁺⁺ transport system, which is modulated by an endogenous calmodulin.     

Identification of calmodulin-like activity in human seminal plasma

Human seminal plasma was found to contain relatively high levels of a heat stable proteinaceous factor with properties similar to that of the calcium-binding protein calmodulin. The seminal plasma factor increases the (Ca²⁺ + Mg²⁺)-ATPase activity found in human red blood cell plasma membranes by 370% and the activation was completely abolished by chlorpromazine, amitriptyline and theophylline. A similar calmodulin-activated Ca²⁺ pump, has been found in the plasma membrane of ram sperm tails. The existence of calmodulin in mammalian seminal plasma may be responsible for some of the metabolic changes associated with sperm maturation.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

Calmodulin can induce and control damped oscillations in plasma membrane Ca<Superscript>2+</Superscript>-ATPase activity: A kinetic model

Plasma membrane Ca²⁺-ATPase is the pump that extrudes calcium ions from cells using ATP hydrolysis to maintain low Ca²⁺ concentrations in the cell. Calmodulin stimulates Ca²⁺-ATPase by binding to the autoinhibitory enzyme domain, which allows the access of cytoplasmic ATP and Ca²⁺ to the catalytic and transport sites. Our kinetic model predicts damped oscillations of the enzyme activity and interprets the known nonmonotonic kinetic behavior of the enzyme in the presence of calmodulin. For parameters close to experimental data, the kinetic model explains the dependence of the frequency and damping factor of the oscillatory enzyme activity on the calmodulin concentration. The calculated pre-steady-state curves fit well to known experimental data. Kinetic analysis allows us to assign Ca²⁺-ATPase to hysteretic enzymes exhibiting activity oscillations in open systems.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

THE IMPORTANCE OF HYDROLYTIC ENZYMES TO AN EXOCYTOTIC EVENT, THE MAMMALIAN SPERM ACROSOME REACTION

The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction. (1) The results of studies utilizing inhibitors of trypsin-like enzymes suggest that such an enzyme has a role in the membrane events of the golden hamster sperm acrosome reaction. The enzyme involved may be acrosin, but it is possible that some as yet unidentified trypsin-like enzyme on the sperm surface may play a role in addition to or instead of acrosin. Results obtained by others with guinea pig, ram and mouse spermatozoa suggest that a trypsin-like enzyme is not involved in the membrane events of the acrosome reaction, but only in the loss of acrosomal matrix. Such results, which conflict with those of the hamster study, may have been due to species differences or the presence of fusion-promoting phospholipase-A or lipids contaminating the incubation media components, and in one case to the possibly damaging effects of the high level of calcium ionophore used. The role of the trypsin-like enzyme in the membrane events of the hamster sperm acrosome reaction may be to activate a putative prophospholipase and/or to hydrolyse an outer acrosomal or plasma membrane protein, thus promoting fusion. A possible role of the enzyme in the vesiculation step rather than the fusion step of the acrosome reaction cannot be ruled out at present. (2) Experiments utilizing inhibitors of phospholipase-A₂, as well as the fusogenic lysophospholipid and cis-unsaturated fatty acid hydrolysis products that would result from such enzyme activity, suggests that a sperm phospholipase-A₂ is involved in the golden hamster sperm acrosome reaction. Inhibitor and LPC addition studies in guinea pig spermatozoa have led others to the same conclusion. The fact that partially purified serum albumin is important in so many capacitation media may be explained by its contamination with phospholipase-A and/or phospholipids. Serum albumin may also play a role, at least in part, by its removal of inhibitory products released by the action of phospholipase-A₂ in the membrane. The demonstration of phospholipase-A₂ activity associated with the acrosome reaction vesicles and/or the soluble component of the acrosome of hamster spermatozoa, and the fact that exogenous phospholipase A₂ can stimulate acrosome reactions in hamster and guinea pig spermatozoa, also support a role for the sperm enzyme. The actual site or the sites of the enzyme in the sperm head are not yet known. The enzyme may be on the plasma membrane as well as, or instead of, in the acrosomal membranes or matrix. A substrate for the phospholipase may be phosphatidylcholine produced by phospholipid methylation. It is possible that more than one type of ‘fusogen’ is released by phospholipase activity (LPC and/or cis-unsaturated fatty acids, which have different roles in membrane fusion and/or vesiculation. In addition to acting as a potential ‘fusogen’, arachidonic acid released by sperm phospholipase-A₂ probably serves as precursor for cyclo-oxygenase or lipoxygenase pathway metabolites, such as prostaglandins and HETES, which might also play a role in the acrosome reaction. Although much evidence points to a role for phospholipase-A₂, phospholipase-C found in spermatozoa could also have a role in the acrosome reaction, perhaps by stimulating events leading to calcium gating, as suggested for this enzyme in somatic secretory cells. (3) A Mg²⁺-ATPase H⁺-pump is present in the acrosome of the golden hamster spermatozoon. Inhibition of this pump by certain inhibitors of ATPases (but not by those that only inhibit mitochondrial function) leads to an acrosome reaction only in capacitated spermatozoa and only in the presence of external K⁺. The enzyme is also inhibited by low levels of calcium, and such inhibition, combined with increased outer membrane permeability to H⁺ and K⁺, and possibly plasma membrane permeability to H⁺ (perhaps by the formation of channels), may be part of capacitation and/or the acrosome reaction. The pH of the hamster sperm acrosome has been shown to become more alkaline during capacitation, and such a change may result in the activation of hydrolytic enzymes in the acrosome or perhaps in a change in membrane permeability to Ca²⁺. A similar Mg²⁺-ATPase has not been found in isolated boar sperm head membranes. However, that conflicting result could have been due to the use of noncapacitated boar spermatozoa for the preparation of the membranes or to protease modification of the boar sperm enzyme during assay. (4) Inhibition of Na⁺, K⁺-ATPase inhibits the acrosome reaction of golden hamster spermatozoa, and the activity of this enzyme increases relatively early during capacitation. A late influx of K⁺ is important for the acrosome reaction. However, this late influx may not be due to Na⁺, K⁺-ATPase, but instead may be due to a K⁺ permeability increase (possibly via newly formed channels) in the membranes during capacitation. It is suggested in this review that Na⁺, K⁺-ATPase has a role early in capacitation rather than directly in the acrosome reaction (although such a role cannot yet be completely ruled out). One possible role for the enzyme in capacitation might be to stimulate glycolysis (which appears to be essential for capacitation and/or the acrosome reaction of hamster and mouse spermatozoa). The function of the influx of K⁺ just before the acrosome reaction is probably to stimulate, directly or indirectly, the H⁺-efflux required for the increase in intraacrosomal pH occurring during capacitation. Direct stimulation of the acrosome reaction by a change in membrane potential resulting directly from K⁺-influx is not a likely explanation for the hamster results. However, the importance of an earlier membrane potential change, due to increased Na⁺, K⁺-ATPase during capacitation, and/or of later membrane potential changes resulting from the pH change, cannot be ruled out. Although K⁺ is required for the hamster acrosome reaction, other workers have reported that K+ inhibits guinea pig sperm capacitation. However, the experimental procedures used in the guinea pig sperm studies raise some questions about the interpretation of those inhibition results. (5) Ca²⁺-influx is known to be required for the acrosome reaction. Others have suggested that increased Ca²⁺-influx due to inhibition or stimulation of sperm membrane calcium transport ATPases are involved in the acrosome reaction. There is as yet no direct or indirect biochemical evidence that inhibition or stimulation of such enzymatic activity is involved in the acrosome reaction, and further studies are needed on those questions. (6) I suggest that the hydrolytic enzymes important to the hamster sperm acrosome reaction will also prove important for the acrosome reaction of all other eutherian mammals.     

The role of calmodulin in the regulation of (Mg2+ + Ca2+)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls

(Mg²⁺ + Ca²⁺)-ATPase activity has been found to be significantly reduced in EDTA-washed erythrocyte membrane preparations from cystic fibrosis patients compared to aged-matched controls. Calmodulin was found to be present in erythrocytes from cystic fibrosis patients and characterized similarly to calmodulin isolated from control preparations. Calmodulin from control erythrocyte preparations stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of EDTA-washed erythrocyte membranes derived from cystic fibrosis patients to the same extent as those membranes derived from controls. Similarly, calmodulin obtained from erythrocytes of cystic fibrosis patients stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of control and cystic fibrosis erythrocyte membrane preparations to a similar extent. These results indicate that this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocytes from cystic fibrosis patients is not due to an alteration in the regulatory function of calmodulin.     

The role of calmodulin in the regulation of (Mg + Ca)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls

(Mg²⁺ + Ca²⁺)-ATPase activity has been found to be significantly reduced in EDTA-washed erythrocyte membrane preparations from cystic fibrosis patients compared to aged-matched controls. Calmodulin was found to be present in erythrocytes from cystic fibrosis patients and characterized similarly to calmodulin isolated from control preparations. Calmodulin from control erythrocyte preparations stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of EDTA-washed erythrocyte membranes derived from cystic fibrosis patients to the same extent as those membranes derived from controls. Similarly, calmodulin obtained from erythrocytes of cystic fibrosis patients stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of control and cystic fibrosis erythrocyte membrane preparations to a similar extent. These results indicate that this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocytes from cystic fibrosis patients is not due to an alteration in the regulatory function of calmodulin.     

钙调蛋白拮抗剂对人胃癌细胞效应机理的研究——钙调蛋白与磷酸二酯酶活性测定分析

我们曾经证明钙调蛋白(Calmodulin,CaM)拮抗剂三氟拉嗪(Trifluoperazine,TFP)有抑制人胃癌细胞增殖和诱导细胞形态向正常分化的效应。本文用CaM活性测定箱方法测定了TFP处理的人胃癌MGC-803细胞胞质内的CaM活性。同时也测定了磷酸二酯酶(Phosphodiesterase.PDE)活性的变化。结果表明TFP选择性抑制胞质内依赖Ca²⁺/CaM的PDE活性。氨茶碱有抑制CaM活化PDE的作用。本文对TFP作用机理及在调控癌细胞增殖及分化中的意义进行讨论。     

Signal transduction pathways in guinea pig sperm

Trifluoperazine (TFP), the antagonist of calmodulin (CaM). significantly stimulated the capacitation and acrosome reaction of guinea pig spermatozoa at the concentration of 10-100μmol/L, independent of the external Ca2+. Forskolin, dbcAMP and caffeine evidently promoted the occurrence of acrosome reaction of spermatozoa at early capacitation stage (5 h) in nonsynchronous system but not in synchronous system. If the spermatozoa were capacitated for 15 h in synchronous system, the above three drugs significantly stimulated acrosome reaction in a Ca2+-independent manner. Protein kinase C activators, i.e. phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) did not influence the occurrence of acrosome reaction of spermatozoa at early capacitation stage, but significantly increased the acrosome reaction rate in capacitated spermatozoa in a Ca2+-independent manner. In contrast. PKC inhibitor staurosporine significantly inhibited the occurrence of acrosome reaction.     

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis

(1) Depending on the assay conditions, the ability of the Ca²⁺-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg²⁺ and K⁺ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca²⁺ so that the activity becomes negligible above 30 μM Ca²⁺. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca²⁺. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca²⁺ are needed and phosphatase activity is maximal in media without Ca²⁺. This is the first report of an activity sustained by the Ca²⁺-ATPase of red cell membranes in the absence of Ca²⁺. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca²⁺. These results strongly suggest that during activation by calmodulin, Ca²⁺ is needed only to form the calmodulin-Ca²⁺ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca²⁺ and the induction of phosphatase activity by ATP + Ca²⁺ suggests that activation of the phosphatase by Ca²⁺ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E₂, the conformer of the Ca²⁺-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca²⁺ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E₁ and E₂ towards E₂. On the other hand, ATP + Ca²⁺ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E₂ predominates over other conformers of the ATPase.     

Calcium-induced protein phosphorylation and changes in levels of calmodulin and calreticulin in maize sperm cells

 To examine possible calcium (Ca²⁺)-mediated prefertilization events in male gametes of higher plants, we studied protein phosphorylation and the Ca²⁺-binding proteins, calmodulin and calreticulin, in sperm cells isolated from maize (Zea mays L.) pollen in the presence and absence of Ca²⁺. Using immunoblotting, we detected calmodulin and calreticulin and Ca²⁺-induced variations. Exposure of sperm cells to 1 mM Ca²⁺ for 1 h increased calmodulin content by 136% compared with the control. Ca²⁺ had little effect on calreticulin at 1 h, but induced a 34% increase after 3 h. Phosphorylation of proteins was low in 1 h-control and Ca²⁺-treated cells. However, a 13-fold increase in phosphorylation of a 18-kDa protein was found at 12 h in the presence of Ca²⁺. Ca²⁺-induced changes in calmodulin, calreticulin and protein phosphorylation observed in maize sperm cells may reflect prefertilization changes in vivo that facilitate sperm cell fusion with egg and central cells. Received: 26 July 1996 / Revision accepted: 7 February 1997     

Calmodulin inhibitors modify cell surface changes triggered by a tumor promoter

Trifluoperazine (TFP) and other inhibitors of the Ca²⁺-binding protein calmodulin, modified two early responses of cultured mammalian cells to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). In the presence of 40μM TFP mouse epidermal cells were insensitive to the TPA-inhibition of epidermal growth factor binding. TFP also caused a marked inhibition of the basal rate of [³H]choline incorporation into HeLa cell phospholipids, and largely overcame the TPA stimulation of choline incorporation.     

Capacitation suppression by mouse seminal vesicle autoantigen involves a decrease in plasma membrane Ca2+‐ATPase (PMCA)‐mediated intracellular calcium

Successful fertilization is tightly regulated by capacitation and decapacitation processes. Without appropriate decapacitation regulation, sperm would undergo a spontaneous acrosome reaction which leads to loss of fertilization ability. Seminal plasma is known to negatively regulate sperm capacitation. However, the suppressive mechanisms still remain unclear. In this study, we demonstrate the decapacitation mechanism of mouse seminal vesicle autoantigen (SVA) might target membrane sphingomyelin (SPM) and regulate plasma membrane Ca²⁺‐ATPase (PMCA) activity. The SVA was shown to suppress sperm capacitation induced by a broad panel of capacitation factors (bovine serum albumin (BSA), PAF, and cyclodextrin (CD)). Furthermore, SVA significantly decreased [Ca²⁺]_i and NaHCO₃‐induced [cAMP]_i. Cyclic AMP agonists bypassed the SVA's suppressive ability. Importantly, the SVA may regulate PMCA activity which was evidenced by the fact that the SVA decreased the [Ca²⁺]_i and intracellular pH (pH_i) of sperm; meanwhile, a PMCA inhibitor (carboxyeosin) could reverse SVA's suppression of [Ca²⁺]_i. The potential target of the SVA on membrane SPM/lipid rafts was highlighted by the high binding affinity of SPM–SVA (with a K_d of ～3 µM) which was close to the IC₅₀ of SVA's suppressive activity. Additionally, treatment of mink lung epithelial cells with the SVA enhanced plasminogen activator inhibitor (PAI)‐1 expression stimulated by tumor growth factor (TGF)‐β and CD. These observations supported the membrane lipid‐raft targeting of SVA. In summary, in this paper, we demonstrate that the decapacitation mechanism of the SVA might target membrane sphingolipid SPM and regulate PMCA activity to lower [Ca²⁺]_i, thereby decreasing the [cAMP]_i level and preventing sperm pre‐capacitation. J. Cell. Biochem. 111: 1188–1198, 2010. © 2010 Wiley‐Liss, Inc.     

Calcium-dependent phosphorylation regulates the plasma-membrane H+-ATPase activity of maize (Zea mays L.) roots

Phosphorylation/dephosphorylation of the plasma-membrane H⁺-ATPase (EC 3.6.1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially purified H⁺-ATPase were used to investigate the effects of Ca²⁺ and calmodulin on the H⁺-ATPase activity and on its phosphorylation status. Both the hydrolytic and the proton-pumping activities were reduced approximately 50% by micromolar Ca²⁺ concentrations while calmodulin did not show any effect either alone or in the presence of Ca²⁺. The lack of effect of calmodulin antagonists indicated that calmodulin was not involved in this response. The addition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca²⁺. Phosphorylation of plasma membrane and partially purified H⁺-ATPase showed the same behavior. In the presence of Ca²⁺ a polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H⁺-ATPase of maize roots. The autoradiogram of the immunodetected protein clearly showed that this polypeptide, which corresponds to the H⁺-ATPase, was phosphorylated. Additional clear evidence comes from the immunoprecipitation experiments: the data obtained show that the H⁺-ATPase activity is indeed influenced by its state of phosphorylation. Received: 19 October 1998 / Accepted: 23 February 1999