Trypsin inhibitors prevent the progesterone-initiated increase in intracellular calcium required for the human sperm acrosome reaction

Inhibitors of trypsin-like enzymes, benzamidine hydrochloride and 4'-acetamidophenyl 4-guanidinobenzoate (also an inhibitor of other serine proteases), were tested for their effects on the acrosome reaction (AR) of human sperm initiated by progesterone or the calcium ionophore ionomycin. The AR was assayed by indirect immunofluorescence and transmission electron microscopy. The trypsin inhibitors, when added 10 min prior to stimulation by progesterone, significantly inhibited the AR in comparison with progesterone treatment alone. Transmission electron microscopic examination of the sperm after progesterone treatment indicated that the inhibitors blocked the membrane fusion events of the AR. By contrast, when ionomycin (at final concentrations of 3 microM) was added to sperm preincubated in inhibitors, sperm underwent morphologically normal AR, acrosomal matrix loss was not inhibited, and the percentage of acrosome-reacted sperm was the same as that obtained in the absence of inhibitors. Using the cell calcium indicator fura-2, we further demonstrated that both trypsin inhibitors prevented the progesterone-stimulated rise in intracellular Ca2+ ([Ca2+]int) required for the AR, but did not affect [Ca2+]int in unstimulated sperm. These results suggest that sperm trypsin-like activity may be directly or indirectly involved in increasing sperm [Ca2+]int during stimulation by progesterone.     

The role of Ca2+ in activation of mature cytotoxic T lymphocytes for lysis

We carried out a detailed analysis of the requirement for Ca2+ in the lysis of target cells by cloned cytotoxic T lymphocytes (CTL). In direct, antigen-specific lysis we always observed an influx of Ca2+ into the CTL concomitant with target cell binding. However, we never observed an increase in CTL Ca2+ content during lectin-mediated lysis, or nonspecific lysis by phorbol myristate acetate-induced CTL. We found that in all three types of lysis (direct, lectin-mediated lysis, C or phorbol myristate acetate-induced) the requirement for Ca2+ in lysis was dictated by the target cell used; the same CTL can kill one target cell in the absence of detectable Ca2+, and absolutely require Ca2+ for the lysis of another target cell. Target cell killing, when it occurred in the absence of Ca2+, was accompanied by microtubule organizing center reorientation in the CTL, showing that this function is not uniformly Ca2+ dependent. These results provide further evidence that Ca2+ is not always required for activation of the lytic pathway in CTL, although Ca2+ may be absolutely required for other CTL functions such as interleukin production or expression of the interleukin 2 receptor.     

Nicotinamide and 3-aminobenzamide interfere with receptor-mediated transmembrane signaling in murine cytotoxic T cells: independence of Golgi reorientation from calcium mobilization and inositol phosphate generation.

The two competitive inhibitors of ADP-ribosylation, nicotinamide and 3-aminobenzamide, have been reported to interfere with TNF-induced cell apoptosis, and there is evidence that they inhibit killer-induced target cell lysis as well. There are very few drugs known to specifically interfere with target apoptosis induced by killer cells. We therefore sought to explore the effects these inhibitors have on CTL-mediated cell lysis. Here we show that TcR-mediated transmembrane signaling in CTL, measured by Ca2+ mobilization and generation of inositol phosphates, is inhibited by nicotinamide. The possibility that all cell functions are suppressed by the drug is excluded by the finding that constitutive secretion of BLT serine esterase is not inhibited, whereas stimulated secretion of this enzyme is suppressed. We also show that nicotinamide does not interfere with CTL target cell binding or reorientation of the Golgi apparatus toward the target binding site. It is concluded that nicotinamide inhibits transmembrane signaling in CTL and thereby interferes with delivery of the lethal hit to targets.     

The mechanism of cell-mediated cytotoxicity. IV. K-76 COONa, which inhibits the activity of Factor I and of C5, inhibits early events in cytotoxic T-lymphocyte-mediated cytolysis and in T-lymphocyte activation

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.     

Different effects of two proteinase inhibitors on insulin-induced cellular responses in rat adipocytes

Among various proteinase inhibitors, N-acetyl-L-tyrosine ethyl ester (ATEE), a chymotrypsin substrate analog, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, showed significant inhibitory effects on insulin stimulated glucose transport in rat adipocytes. ATEE did not affect insulin binding, but inhibited insulin internalization. In intact adipocytes, ATEE inhibited tyrosine phosphorylation of the beta-subunit of the insulin receptor, a 170 kDa protein and a 60 kDa protein at almost the same concentration (ID50 = 0.24 +/- 0.05 mM, n = 4, mean +/- S.E.), but in a plasma membrane fraction, ATEE did not appreciably inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor, TLCK did not inhibit insulin binding. At 0.25 mM, TLCK did not inhibit insulin internalization, but inhibited 70% of the insulin-stimulated glucose transport (ID50 = 0.19 +/- 0.02 mM, n = 7). TLCK inhibited insulin internalization at more than 0.25 mM. TLCK did not inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor in intact cells or in the plasma membrane fraction. In intact cells, TLCK inhibited the phosphorylation of the 60 kDa protein and simultaneously it stimulated the phosphorylation of the 170 kDa protein more than 3-fold. These results indicate that there are at least two sites in the insulin-induced signal transduction pathway where proteinase inhibitors act to suppress the insulin signal transduction. A major ATEE site is very close to phosphorylation of the beta-subunit of the insulin receptor. On the other hand, TLCK inhibits a step(s) in the signal transduction pathway after the insulin receptor but before the glucose transporter.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

Inhibition of cytotoxic T lymphocyte-mediated lysis and cellular proliferation by isoquinoline sulfonamide protein kinase inhibitors. Evidence for the involvement of protein kinase C in lymphocyte function

The effects of the isoquinoline sulfonamides, a class of synthetic protein kinase inhibitors, namely 1-(5-isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride (H7), N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H8), N-(2-aminoethyl)-5-isoquinoline sulfonamide dihydrochloride (H9), and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA1004), on the lytic activity of in vivo-produced (H-2b anti-H-2d alloimmune) cytotoxic T lymphocytes (CTL) were investigated. The hierarchy of inhibition of lysis shown by these compounds resembled that of their inhibition of Ca2+/phospholipid-dependent enzyme (protein kinase C). H7 has the highest affinity for protein kinase C (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041) and gave the greatest inhibition of lysis by CTL. HA1004 has the weakest affinity for protein kinase C and gave very little inhibition of lysis, whereas H8 and H9 showed intermediate inhibition of lysis. In addition, the effect of the isoquinoline sulfonamides on cellular proliferation was examined. Interestingly, the pattern of inhibition observed for both lymphocytes and tumor cells closely mimicked the effects of these compounds on protein kinase C activity. These results demonstrate that modulation of an early biochemical signal affects both short-term (e.g. CTL-mediated lysis) and long-term (e.g. cellular proliferation) events. These data provide further evidence for the integral role of protein kinase C in the activation of the lytic signal in CTL. In addition, suggestive evidence is provided that protein kinase C, or some other enzyme with similar sensitivity to the isoquinoline sulfonamides, plays an important role in cellular proliferation.     

Differential effects of protein synthesis inhibition on CTL and targets in cell-mediated cytotoxicity

The reactions that lead to target cell lysis by cytotoxic T cells (CTL) are despite intensive investigations poorly understood. To examine the relative roles effectors and targets play in the lytic reaction, protein synthesis in either CTL or targets was inhibited before assay of lysis. We show, in agreement with previous results, that de novo protein synthesis is not necessary in either effectors or targets during the cytolytic reaction. However, activation of CTL requires protein synthesis. Activated CTL respond to protein synthesis inhibitors with a cycling of activity, a result that is interpreted to be consistent with a stimulus secretion mechanism. Treatment of targets with protein synthesis inhibitors prior to incubation with CTL leads to a very rapid and irreversible loss of lytic susceptibility. It is shown that the decrease in lysability is not due to lack of proper CTL target interaction: MHC class I antigens are expressed on drug-treated targets and these cells serve as cold targets in competitive inhibition experiments. Moreover, drug-treated targets trigger transient Ca2+ mobilization and generation of inositol phosphates in CTL. It is therefore concluded that drug-treated targets are able to trigger CTL function but lack a component that is required for their successful lysis.     

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes. These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.     

Cytotoxic T lymphocyte-induced loss of target cell adhesion and lysis involve common and separate signaling pathways

Recently, we demonstrated that an early event in the CTL-target cell (TC) interaction is loss of TC adherence to substrate. This loss of adhesion is Ag-specific, but distinct from the lytic event because it can ensue in nominally Ca2+-free medium. In this study, we examine further the mechanism of CTL-induced loss of adhesion, concentrating mainly on the signal transduction pathway. Based on the differential sensitivity of CTL to extracellular Ca2+, protein kinase C activation/depletion and inhibition by anti-Lyt-2 (CD8) or anti-CTL receptor (TCR) reagents, we demonstrate that CTL-induced loss of adhesion can be initiated through multiple activation pathways. Although CTL-mediated lysis is restricted to a Ca2+ and protein kinase C-dependent signaling mechanism, CTL-induced loss of adhesion is initiated in the presence or absence of extracellular Ca2+ or functional protein kinase C activity. Furthermore, although under physiologic conditions, anti-CD8 or anti-TCR reagents strongly block both CTL activities, under non-lytic conditions, they fail to inhibit the ability of CTL to promote loss of adhesion. These findings implicate the participation of additional CTL-TC ligand interactions resulting in loss of adhesion, and thus, provide further evidence to support the hypothesis that CTL-induced loss of adhesion can be initiated through multiple triggering pathways.     

Maturation of cytolytic T lymphocytes

We have studied the maturation of cytotoxic T-lymphocytes (CTL) following primary and anamnestic responses in vivo and in vitro. Parameters evaluated included: frequency of effector CTL, specificity of binding to and lysis of target cells, killing and recycling ability of individual CTL, and the avidity of effector-target conjugation. While the frequency of effector CTL in the peritoneal cavity of BALB/c mice immunized against leukemia EL4 of C57BL/6 origin increases from 0 to 35% in 11 days of priming, a paradoxically lower frequency has been observed usually after 2 degrees and repeatedly after 3 degrees immunizations both in the peritoneal cavity and in the spleen. The H-2 haplotype and H-2 sub-loci specificity of CTL is preserved upon repeated immunizations. Likewise, the rate of killing and recycling of individual CTL do not change throughout immunizations, suggesting that the cytolytic activity of individual effector CTL is discrete ("quantal") and not subject to maturation upon repeated immunizations. On the other hand, inhibition of conjugate formation and of lysis by antibodies against target major histocompatibility complex (MHC) or effector Lyt-2 determinants is consistently less effective with 3 degrees CTL, suggesting an increase in avidity of effector/target interaction upon repeated immunizations. A striking increase in apparent avidity has been observed during CTL priming in mixed lymphocyte reaction, as deduced from blocking by target cell MHC antibodies. These results suggest that alloimmune CTL undergo maturation with respect to their ability to interact with the target, and that the composition of the responding population is subject to moderate selective processes driven by repeated antigenic stimuli.     

Inhibition of adenosine kinase and adenosine uptake in guinea-pig CNS tissue by halogenated tubercidin analogues

Two halogenated analogues of tubercidin (7-deazaadenosine) viz. 5-iodotubercidin and 5′-deoxy-5-iodotubercidin, previously were shown to be potent inhibitors of guinea-pig brain adenosine kinase activity and adenosine uptake in guinea-pig cerebral cortex slices. A further series of halogenated tubercidin analogues have been investigated; of the 9 compounds tested, 5′-deoxy-5-iodotubericidin was the most potent adenosine kinase inhibitor while 5-iodotubercidin was the most potent in inhibiting the facilitated uptake of adenosine. These compounds may be useful for elucidating the involvement of adenosine kinase in adenosine uptake, the maintenance of intracellular adenosine levels and in the neuromodulatory actions of adenosine in the CNS.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

Evidence for multiple lytic pathways used by cytotoxic T lymphocytes

Previous data generated by ourselves and others questioned the role of degranulation as a mechanism to explain CTL-mediated cytotoxicity. In this report we examine this tissue in greater depth. CTL-mediated lysis was probed with three different inhibitors. 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene inhibits degranulation in a wide range of cell types, including CTL. EGTA, through chelation of Ca2+, also inhibits degranulation processes in CTL, and would inhibit other events or processes dependent on extracellular Ca2+. We also used prolonged exposure to PMA to exhaust PKC activity in CTL. Using these inhibitors, we have defined three pathways of lysis used by CTL. One pathway requires Ca2+, is PMA sensitive, but does not depend on degranulation. The second pathway is independent of Ca2+, is not PMA sensitive, and also does not depend on degranulation. All primary CTL and cloned CTL lyse most target cells via pathway I. However, when confronted with certain target cells (which we have referred to previously as Ca2+-independent target cells), pathway II is induced. When pathway II is induced, pathway I apparently shuts down. We show here that pathway II does not depend on protein synthesis, and that it also leads to DNA solubilization in target cells. A limited number of cloned CTL use pathways I and II as just described, but use in addition, and simultaneously, a third pathway that appears to involve degranulation. This pathway is seen irregularly in most CTL clones, and may be influenced by levels of IL-2 in the culture medium.     

T lymphocyte-mediated cytolysis: dissociation of the binding and lytic mechanisms of the effector cell.

To determine functional relationships between the cytotoxic T lymphocyte (CTL) receptor for target binding and the lytic mechanism, we have studied the reaction between two immunized CTL populations (AalphaB and BalphaA), both at the population and the single-cell level. When studied at the population level, the reaction of AalphaB with BalphaA (bidirectional system) resulted in formation of AalphaB/BalphaA conjugates and bidirectional cytolysis. However, when the viability of cells in individual AalphaB/BalphaA conjugates was analyzed, unidirectional instead of bidirectional lysis occurred. These results indicate that under conditions that are conducive to lysis, binding of a potentially lytic cell to its target does not necessarily result in target lysis. Short heat treatment of CTL (44 degrees C, 10 min) totally abolished their lytic activity, without affecting their capacity to bind specifically, thus dissociating the binding from the lytic activity of the CTL. The cytolytic activity is probably associated with, or triggered by the CTL-binding unit. The binding unit, on the other hand, appears to be a functional receptor of the CTL, which is involved in but not sufficient to bring about lysis.     

Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.     

Characterization of the pharmacological-sensitivity profile of neoglycoprotein-induced acrosome reaction in mouse spermatozoa.

Mammalian spermatozoa undergo the acrosome reaction (AR) in response to the interaction of a carbohydrate-recognizing molecule(s) on the sperm plasma membrane (sperm surface receptor) and its complementary glycan (ligand) moiety(ies) on the zona pellucida (ZP). Previously, we demonstrated that a hexose (mannose) or two amino sugars (glucosaminyl or galactosaminyl residues) when covalently conjugated to a protein backbone (neoglycoproteins) mimicked the mouse ZP3 glycoprotein and induced the AR in capacitated mouse spermatozoa (Loeser and Tulsiani, Biol Reprod 1999; 60:94-101). To elucidate the mechanism underlying sperm-neoglycoprotein interaction and the induction of the AR, we have examined the effect of several AR blockers on neoglycoprotein-induced AR. Our data demonstrate that two known L-type Ca(2+) channel blockers prevented the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA). The fact that the L-type Ca(2+) channel blockers (verapamil, diltiazem) had no inhibitory effect on sperm surface galactosyltransferase or alpha-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.     

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.     

A diversity of SERCA Ca2+ pump inhibitors

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.