Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa

New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H₂O₂ and O₂ production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24h, 48h and 72h.Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24h, 48h and 72h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.     

Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa

The aim of this study was to determine the localization of calmodulin (CaM) in ram sperm and the possible changes during in vitro capacitation (CA) and the ionophore-induced acrosome reaction (AR). Likewise, changes in intracellular calcium levels ([Ca²⁺]_i) were also analysed by using flow cytometry. CA was induced in vitro in a medium containing BSA, CaCl₂, NaHCO₃, and AR by the addition of the calcium ionophore A23187. The acrosomal status was assessed by the chlortetracycline-fluorescence (CTC) assay. Flow cytometry (FC) analyses were performed by loading samples with Fluo-3 AM, that emits fluorescence at a high [Ca²⁺]_i, combined with propidium iodide (PI) that allowed us to discriminate sperm with/without an integral plasma membrane both with high/low [Ca²⁺]_i. Immunocytochemistry localized CaM to the flagellum, and some sperm also contained CaM in the head (equatorial and post-acrosomal regions). CA and AR resulted in a slight increase in the post-acrosomal labelling. The treatment of sperm with increasing concentrations of two CaM antagonists, W7 and calmidazolium (CZ), accounted for an increase in capacitated and acrosome-reacted CTC-sperm patterns. CZ induced a significant reduction in the content of three protein tyrosine-phosphorylated bands of approximately of 30, 40 and 45 kDa. However, W7 showed no significant effect at any of the studied concentrations. Neither of them significantly influenced protein serine and threonine phosphorylation. FC analysis revealed that the main subpopulation in the control samples contained 70% of the total sperm with integral plasma membrane and a medium [Ca²⁺]_i. After CA, 67.1% of the sperm preserved an integral membrane with a higher [Ca²⁺]_i. After AR, only 7.2% of the total sperm preserved intact membranes with a very high [Ca²⁺]_i. These results imply that CaM appears to be involved in ram sperm capacitation, and both treatments increased its localization in the post-acrosomal region.     

Effects of frozen and liquid hypothermic storage and extender type on calcium homeostasis in relation to viability and ATP content in striped bass (Morone saxatilis) sperm

The effect of hypothermic storage on striped bass sperm calcium homeostasis was determined by Fluo-3 flow cytometry. Calcium homeostasis was defined as the ability of cells to maintain a low concentration of intracellular free calcium as measured by Fluo-3 fluorescence. Sperm were stored frozen in striped bass extender (SBE) and Tris–NaCl medium (T350) modified with 50 mM glycine and 7.5% dimethylsulfoxide and in nonfrozen form diluted 1:3 (vol/vol) in SBE and T350 for 1, 24, and 48 hours at 4 °C in an oxygen atmosphere. Fluo-3 fluorescence was detected in less than 5% of fresh viable sperm cells indicating maintenance of calcium homeostasis. In contrast to sperm in fresh semen, frozen-thawed and nonfrozen sperm cells lost to a considerable extent the ability to maintain low intracellular free calcium even in the absence of exogenous calcium; positive Fluo-3 fluorescence was found in 26% and 39% of thawed sperm frozen in SBE- and T350-based freezing diluents, respectively, and increased (P < 0.05) to 67% during nonfrozen storage in SBE and T350 at 24 and 48 hours. Sperm viability measured by exclusion of propidium iodide by flow cytometry was 99% in fresh milt and maintained at 86% (P > 0.05) in SBE after 48 hours of nonfrozen storage but decreased (P < 0.05) to 55.7% after 48 hours in T350. Energy status in terms of ATP content, determined by luciferin–luciferase bioluminescence assay, was higher (P < 0.05) in sperm frozen in SBE than in T350 during the first 5 minutes post-thaw and decreased to essentially zero by 15 minutes post-thaw and did not differ among nonfrozen storage treatments. In conclusion, sperm cells impervious to propidium iodide after frozen or nonfrozen storage were unable to maintain low intracellular calcium content. SBE is a better medium than T350 for frozen or nonfrozen storage of striped bass sperm. The inability to regulate intracellular calcium in striped bass sperm may be associated with poor activation of motility after 4 °C storage and cryopreservation.     

Proteolytic processing of phospholipase Czeta and [Ca2+]i oscillations during mammalian fertilization

Phospholipase Cζ (PLCζ) is a sperm-specific PLC capable of causing repetitive intracellular Ca²⁺ ([Ca²⁺]_i) release ([Ca²⁺]_i oscillations) in mammalian eggs. Accumulating evidence suggests that PLCζ is the sperm factor responsible for inducing egg activation. Nevertheless, some sperm fractions devoid of 72-kDa PLCζ showed [Ca²⁺]_i oscillation-inducing and PLCζ-like PLC activity (Kurokawa et al., (2005) Dev. Biol. 285, 376-392). Here, we report that PLCζ remains functional after proteolytic cleavage at the X-Y linker region. We found that N-terminal (33 and 37 kDa) and C-terminal fragments (27 kDa), presumably the result of PLCζ cleavage at the X-Y linker region, were present in fresh sperm as well as in sperm extracts and remained associated as functional complexes. Protease V8 cleaved 72-kDa PLCζ into 33/37 and 27 kDa fragments, while PLC activity and [Ca²⁺]_i oscillation-inducing activity persisted until degradation of the fragments. Immunodepletion or affinity depletion of these fragments abolished PLC activity and [Ca²⁺]_i oscillation-inducing activity from sperm extracts. Lastly, co-expression of cRNAs encoding residues 1-361 and 362-647 of mouse PLCζ, mimicking cleavage at the X-Y linker region, induced [Ca²⁺]_i oscillations and embryo development in mouse eggs. Our results support the hypothesis that PLCζ is the sole mammalian sperm factor and that its linker region may have important regulatory functions during mammalian fertilization.     

The Involvement of PI3K-Mediated and L-VGCC-Gated Transient Ca2+ Influx in 17β-Estradiol-Mediated Protection of Retinal Cells from H2O2-Induced Apoptosis with Ca2+ Overload

Intracellular calcium concentration ([Ca²⁺]_i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca²⁺]_i and its control mechanisms in process of hydrogen peroxide (H₂O₂)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca²⁺ indicator to detect [Ca²⁺]_i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H₂O₂-induced retinal cell apoptosis occurred at 4 h after H₂O₂ stress and increased in a time-dependent manner, but [Ca²⁺]_i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H₂O₂ stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca²⁺]_i, which appeared only at 0.5 h after βE2 application; c) increased [Ca²⁺]_i under 100 µM H₂O₂ treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca²⁺ stores; d) importantly, the transiently increased [Ca²⁺]_i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca²⁺ channels (L-VGCC), but the increased [Ca²⁺]_i induced by 100 µM H₂O₂ treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H₂O₂, which was also associated with its transient [Ca²⁺]_i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.     

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background

We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca²⁺ [Ca²⁺]_i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca²⁺]_i.

Methods

HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.

Results

Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl₂ on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca²⁺ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl₂ when tested after cryostorage. These functional effects were reflected by changes in [Ca²⁺]_i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.

Conclusions

Data suggested that cryopreservation modified [Ca²⁺]_i of venous smooth muscle cells (1) through depolarization-induced changes in Ca²⁺ influx and (2) through activation of Rho kinase signaling pathways.     

Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.     

Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O₂⁻) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O₂⁻ level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.     

Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization,Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells

Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca²⁺]_i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca²⁺]_i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca²⁺]_i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca²⁺]_i increases from histamine, but failed to inhibit [Ca²⁺]_i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca²⁺]_i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca²⁺]_i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca²⁺]_i inhibition and TAS2R subtype, nor whether [Ca²⁺]_i was increased by G_q- or G_i-coupled GPCRs. Selected studies revealed a correlation between [Ca²⁺]_i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca²⁺]_i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca²⁺]_i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca²⁺]_i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca²⁺]_i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca²⁺]_i inhibition limb.     

Properties of a Novel pH-dependent Ca2+ Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function

Rises of intracellular Ca²⁺ ([Ca²⁺]_i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca²⁺]_i and intracellular pH (pH_i) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca²⁺ channels modulated by pH_i in the origin of these Ca²⁺ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca²⁺]_i on identified spermatogenic cells from mouse adult testes. In BCECF [(2′,7′)-bis(carboxymethyl)- (5,6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30–60 s) application of 25 mM NH₄Cl increased pH_i by ∼1.3 U from a resting pH_i ∼6.65. A steady pH_i plateau was maintained during NH₄Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca²⁺]_i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca²⁺ influx or intracellular Ca²⁺ release demonstrated that the majority of the Ca²⁺ rise results from plasma membrane Ca²⁺ influx, although a small component likely to result from intracellular Ca²⁺ release was occasionally observed. Ca²⁺ transients potentiated with repeated NH₄Cl applications, gradually obliterating the initial [Ca²⁺]_i drop. The pH-sensitive Ca²⁺ permeation pathway allows the passage of other divalents (Sr²⁺, Ba²⁺, and Mn²⁺) and is blocked by inorganic Ca²⁺ channel blockers (Ni²⁺ and Cd²⁺), but not by the organic blocker nifedipine. The magnitude of these Ca²⁺ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca²⁺ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca²⁺ channel also present in spermatogenic cells. We postulate that the Ca²⁺ permeation pathway regulated by pH_i, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca²⁺ influx required for initiating the acrosome reaction and perhaps other important sperm functions.     

Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Opioid-Induced Increase in [Ca2+]i in ND8-47 Neuroblastoma × Dorsal Root Ganglion Hybrid Cells Is Mediated Through G Protein-Coupled δ-Opioid Receptors and Desensitized by Chronic Exposure to Opioid

Abstract: δ-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca²⁺]_i) in ND8-47 cells by activating dihydropyridine-sensitive Ca²⁺ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d -Ser²,Leu⁵]enkephalin-Thr (DSLET)-induced increase in [Ca²⁺]_i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca²⁺]_i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [³H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits α_i2, α_i3, α_s, and α_q were detected in ND8-47 cell membranes by western blot; α_o and α_i1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α-subunits. These results suggest that the DSLET-induced increase in [Ca²⁺]_i is mediated through pertussis toxin-sensitive G proteins (probably G_i2 or G_i3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

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.     

Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa

The effects of different dilutions of seminal plasma (SP) on the qualitative characteristics of rabbit spermatozoa and on their fertilising ability were analysed. Ejaculated semen was centrifuged twice and the sperm resuspended in media with decreasing ratios of SP/Tris: (1/2; 1/5; 1/10; 1/20; 1/30; 1/100) until the complete substitution was with SP. The control constituted sperm in undiluted SP. Samples were maintained at 37 degrees C and kinetic analysis done at fixed intervals (0-6h). Also the thiobarbituric reactive substances (TBA-RS) values were determined. Rabbit sperm suspended in Tris, or with extremely low content of SP, lost motility and viability within 1-3h, while sperm suspended in SP either undiluted or diluted up to 10-fold, showed similar motility during the 6h period (from 39 to 49%). Further dilutions of SP (1/20-1/30) had no effect during the initial 2h of storage but thereafter the decline of motility was more marked (after 6h: from 0 to 17%). Kinetic parameters followed the same trend and differences were particularly marked after storage: the highest values were in samples diluted up to 1/10; a sharp decline in motility characteristics was observed at higher dilutions. The addition of SP (1/2 v/v) to immotile sperm reactivated 35.5% of cells. However, SP did not significantly affect fertility rate or litter size possible involving an interaction with the female reproductive tract. SP reduced lipid oxidation (TBA-RS) of semen only after storage. A positive correlation between final TBA-RS and cell viability indicated that peroxidation was one of the cause of rabbit sperm deterioration during conservation.