Increase In Intracellular Calcium Concentration Research Articles

Prostaglandin A1 inhibits increases in intracellular calcium concentration, TXA2 production and platelet activation1

In our previous studies we found that cyclopentenane prostaglandin A1 (PGA1) had neuroprotective effects in a rodent ischemic model. In the present study we aimed to investigate the inhibitory effect of PGA1 on platelet function. The rate of aggregation of human platelets was measured by using turbidimetry. The rate of adhesion of platelets to cultured endothelial cells was determined by using [(3)H]-adenine labeled platelets. 5-Hydroxytryptamine release from platelets was measured with O-phthaldialdehyde fluorospectrophotometry. The levels of TXB(2), a stable metabolite of TXA(2), were determined by radioimmuno-assay. Alternations in platelet morphology were observed using an electron microscope, and the intraplatelet free calcium concentrations were measured with Fluo-3/AM FCM assay. PGA1 significantly inhibited thrombin-, collagen- and ADP-induced aggregation and adhesion of platelets. The morphological changes of platelets induced by thrombin were blocked by PGA1. PGA1 inhibited the release of 5-hydroxytyptamine from dense granules and the synthesis of TXA(2). PGA1 inhibits the activation of platelets probably through blocking increases in intracellular calcium concentration and TXA(2) synthesis.

PKCα Mediates CCL18-Stimulated Collagen Production in Pulmonary Fibroblasts

A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCalpha, PKCdelta, and PKCepsilon), two isoforms (PKCdelta and PKCepsilon) have been implicated in profibrotic intracellular signaling. The role of PKCalpha-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCalpha was found mostly in the cytoplasm, whereas PKCdelta and PKCepsilon were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCalpha but not PKCdelta or PKCepsilon underwent rapid (within 5-10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCalpha and ERK2, but not PKCdelta or PKCepsilon, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCalpha Gö6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCalpha signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCalpha nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCalpha but not PKCdelta or PKCepsilon mediate the profibrotic effect of CCL18. PKCalpha may therefore become a viable target for future antifibrotic therapies.

ATP‐induced apoptotic cell death in porcine ovarian theca cells through P2X7 receptor activation

Folliculogenesis modulation via distinct neurotransmitters is a well-documented phenomenon. Intraovarian purinergic signaling mechanisms have been identified previously in different species. However, the molecular elements involved and the physiological role of this purinergic signaling remain to be elucidated. Here, studies using RT-PCR amplification, immunoblotting, and immunofluorescence microscopy showed that murine and porcine ovaries express the P2X7 subtype receptor, a cationic receptor-channel operated by ATP. Using immunofluorescence it was demonstrated that P2X7 protein expression, in both mouse and pig, occurs specifically in the theca cells from antral follicles. Isolated porcine theca cells maintained in primary cultures and tested with 1 mM ATP or 250 microM Bz-ATP, a specific agonist of P2X7, responded with an increase in intracellular calcium concentration, as demonstrated in cells loaded with fluo-4 as calcium indicator. This strongly suggested that P2X7 receptors in theca cells are functional. Moreover, application for 24 hr of 1 mM ATP or 250 microM Bz-ATP induced apoptotic cell death as indicated by the DNA fragmentation pattern, positive TUNEL test, and annexin V binding. This ATP effect was antagonized by 300 microM PPADS and 200 microM oxidized ATP. Also, addition of 5 mM EGTA in the external medium to chelate free Ca++ decreased death cell to 24% of that produced by 200 microM Bz-ATP, suggesting that Ca++ influx participates in the phenomenon. The highly specific and functional expression of P2X7 receptors in theca cells suggest a role for ATP in modulating follicular physiology.

Gene expression is differentially regulated by neurotransmitters in embryonic neuronal cortical culture

Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

Calcium Turns a Blind Eye to an Unchaperoned Photoreceptor

Rosenbaum et al. have uncovered an intriguing duality of function for calnexin, a calcium-binding molecular chaperone with an N-terminal domain in the lumen of the endoplasmic reticulum (ER) and a C-terminal tail in the cytosol. The authors found that two independent Drosophila mutations in the coding region of calnexin ( cnx ) were associated with a decrease in the abundance of rhodopsin (Rh1), the G protein-coupled receptor that activates the visual transduction pathway. The expression of other photoreceptor proteins [including Rh3, Rh4, and Rh5 opsins; chaoptin; the TRP and TRPL channels; phospholipase C-β (PLC-β); arrestins 1 and 2; and Gαq] was not affected. Although the abundance of RH1 transcripts was not affected, RH1 maturation was impaired. Cnx formed a stable complex with Rh1, and immunocytochemical analysis confirmed that, in Drosophila photoreceptors, Cnx localized to the ER. Cnx mutants also showed age-dependent retinal degeneration, which was enhanced by light and slowed by mutation of PLC-β. PLC-β, which is activated during the phototransduction cascade, stimulates the opening of TRP and TRPL, thereby eliciting the influx of calcium, which can be toxic at high concentrations. Calcium imaging revealed that the increase in intracellular calcium concentration in response to illumination was markedly greater in cnx mutant photoreceptor cells than in wild-type cells or cells with a mutation in another chaperone required for Rh1 maturation that does not have a calcium-binding domain. Thus, the authors conclude that Cnx plays a dual role in photoreceptor cells, serving not only as a Rh1 chaperone but also as a calcium buffer. E. E. Rosenbaum, R. C. Hardie, N. J. Colley, Calnexin is essential for rhodopsin maturation, Ca 2+ regulation, and photoreceptor cell survival. Neuron 49 , 229-241 (2006). [PubMed]

Different Conformations Prefer Distinct Signaling Pathways

The metabotropic glutamate receptor 1α (mGluR1α), a homodimeric G protein-coupled receptor critically involved in some forms of synaptic plasticity, can stimulate an increase in intracellular calcium concentration ([Ca 2+ ] i ) through Gq or an increase in intracellular adenosine 3′,5′-monophosphate (cAMP) concentration ([cAMP] i ) through Gs. Each mGluR1α subunit contains an extracellular "Venus flytrap" module; glutamate binding stabilizes an active conformation in which the Venus flytrap of one subunit closes while the other stays open (closed-open/active, CO/A). In the presence of Gd 3+ , which binds to the interface between subunits, glutamate binds to both subunits, leading to a closed-closed/active (CC/A) conformation. Tateyama and Kubo used fluorescent indicators to simultaneously measure [Ca 2+ ] i and [cAMP] i in CHO (Chinese hamster ovary) cells expressing mGluR1α and explored the relation between the two active conformations and coupling to the two signaling pathways. Whereas glutamate stimulated increases in both [Ca 2+ ] i and [cAMP] i , Gd 3+ stimulated an increase only in [Ca 2+ ] i . Moreover, Gd 3+ decreased mGluR1α activation of Gs (the ability to increase [cAMP] i ) in response to glutamate but did not affect coupling to Gq (the ability to increase [Ca 2+ ] i ). Thus, the authors conclude that, rather than acting as a simple "on/off switch," the different active conformations of mGluR1α preferentially interact with distinct G proteins and thereby activate distinct intracellular signaling pathways. M. Tateyama, Y. Kubo, Dual signaling is differentially activated by different active states of the metabotropic glutamate receptor 1α. Proc. Natl. Acad. Sci. U.S.A. 103 , 1124-1128 (2006). [Abstract] [Full Text]

Heterogeneous response of microvascular endothelial cells to shear stress

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.

Regulation of Volume-Sensitive Osmolyte Efflux from Human SH-SY5Y Neuroblastoma Cells following Activation of Lysophospholipid Receptors

The ability of the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) to promote the release of the organic osmolyte taurine in response to hypoosmotic stress has been examined. Incubation of SH-SY5Y neuroblastoma cells under hypoosmotic conditions (230 mOsM) resulted in a time-dependent release of taurine that was markedly enhanced (3-7-fold) by the addition of micromolar concentrations of either S1P or LPA. At optimal concentrations, the effects of S1P and LPA on taurine efflux were additive and mediated via distinct receptors. Inclusion of 1,9-dideoxyfoskolin, 5-nitro-2-(3-phenylpropylamino benzoic acid, or 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]-butanoic acid blocked the ability of both lysophospholipids to enhance taurine release, indicating the mediation of a volume-sensitive organic osmolyte and anion channel. Both S1P and LPA elicited robust increases in intracellular calcium concentration that were attenuated by the removal of extracellular calcium, abolished by the depletion of intracellular calcium with thapsigargin, and were independent of phosphoinositide turnover. Taurine efflux mediated by S1P and LPA was unaffected by the removal of extracellular calcium but was attenuated by depletion of intracellular calcium (34-38%) and by inhibition of protein kinase C (PKC) with chelerythrine (38-72%). When intracellular calcium was depleted and PKC was inhibited, S1P- or LPA-stimulated taurine efflux was inhibited by 80%. Pretreatment of the cells with pertussis toxin, toxin B, or cytochalasin D had no effect on lysophospholipid-stimulated taurine efflux. The results indicate that both S1P and LPA receptors facilitate osmolyte release via a phospholipase C-independent mechanism that requires the availability of intracellular calcium and PKC activity.

Wine polyphenols stimulate superoxide anion production to promote calcium signaling and endothelial-dependent vasodilatation.

The present study was aimed to evaluate the mechanisms involved in the vasorelaxant effects of red wine polyphenol compounds (RWPC) in small mesenteric rat arteries. RWPC produce relaxation in small mesenteric arteries. This relaxant effect was abolished by endothelial denudation, NO-synthase blockade with L-NAME and partial depolarization with KCl or L-NAME plus KCl. Incubation with the reactive oxygen species scavenger, superoxide dismutase (SOD) plus catalase, or inhibition of NAD(P)H-dependent oxidoreductases with diphenyleneiodonium also inhibited RWPC induced vascular relaxation. Application of RWPC elicited a transient increase in intracellular calcium concentration ([Ca2+]i) in bovine aortic endothelial cells (BAEC), which was attenuated by a mixture of SOD and catalase. Incubation of BAEC with RWPC increased the SOD inhibitable production of O2-. These results suggest the involvement of O2- in the [Ca2+]i increase evoked by RWPC, leading to the activation of enzymes involved in the release of endothelial relaxant factors and subsequent vasodilatation of resistance arteries.

Protective effect of donepezil in primary-cultured rat cortical neurons exposed to N-methyl- d-aspartate (NMDA) toxicity

Donepezil has a neuroprotective effect against oxygen–glucose deprivation injury and glutamate toxicity in cultured cortical neurons. In this study, we further characterized the neuroprotective properties of donepezil in rat cortical cell cultures using glutamate receptor-specific agonists ( N-methyl- d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate). Pretreatment with donepezil (1 μM) for 12 h significantly decreased the lactate dehydrogenase (LDH) release in response to NMDA (100 μM) by 43.8%, and reduced the LDH release in response to kainate (100 μM) and AMPA (100 μM) by 11.9% and 7.5% (without statistical significance), respectively. Donepezil appeared to inhibit LDH release in a concentration-dependent manner at 0.1–10 μM. Cortical neurons exposed to NMDA retained a normal morphological appearance in the presence of 10 μM donepezil. In binding assay for glutamate receptors, donepezil at 100 μM only slightly inhibited binding to the glycine and polyamine sites on NMDA receptor complex. On the other hand, 12 h pretreatment with donepezil at 10 and 100 μM significantly decreased the NMDA-induced increase of intracellular calcium concentration ([Ca 2+] i). In conclusion, our results show that donepezil has protective activity against NMDA toxicity in cortical neurons, and this neuroprotection seems to be partially mediated by inhibition of the increase of [Ca 2+] i.

Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

Water Channels and Zymogen Granules in Salivary Glands

Salivary secretion occurs in response to stimulation by neurotransmitters released from autonomic nerve endings. The molecular mechanisms underlying the secretion of water, a main component of saliva, from salivary glands are not known; the plasma membrane is a major barrier to water transport. A 28-kDa integral membrane protein, distributed in highly water-permeable tissues, was identified as a water channel protein, aquaporin (AQP). Thirteen AQPs (AQP0 - AQP12) have been identified in mammals. AQP5 is localized in lipid rafts under unstimulated conditions and translocates to the apical plasma membrane in rat parotid glands upon stimulation by muscarinic agonists. The importance of increases in intracellular calcium concentration [Ca(2+)](i) and the nitric oxide synthase and protein kinase G signaling pathway in the translocation of AQP5 is reviewed in section I. Signals generated by the activation of Ca(2+) mobilizing receptors simultaneously trigger and regulate exocytosis. Zymogen granule exocytosis occurs under the control of essential process, stimulus-secretion coupling, in salivary glands. Ca(2+) signaling is a principal signal in both protein and water secretion from salivary glands induced by cholinergic stimulation. On the other hand, the cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase system has a major role in zymogen granule exocytosis without significant increases in [Ca(2+)](i). In section II, the mechanisms underlying the control of salivary protein secretion and its dysfunction are reviewed.

Serotonin increases protective duodenal bicarbonate secretion via enteric ganglia and a 5-HT4-dependent pathway

Objective. Serotonin (5-HT) is present in much larger amounts in the gut than in the central nervous system and is predominantly synthesized and stored in mucosal enterochromaffin cells. Bicarbonate secretion by the duodenal mucosa is the major mechanism in maintaining mucosal integrity, neutralizing invading protons within the surface mucus gel. In this study the role of local 5-HT in the control of the protective secretion was investigated. Material and methods. A segment of proximal duodenum was perfused in situ in anaesthetized rats and the alkaline secretion was continuously recorded by pH-stat. Intracellular calcium signalling was measured in clusters of human and rat duodenal enterocytes devoid of neural tissue. After loading with the fluorescent probe, fura-2, the clusters were attached to the bottom of a temperature-controlled perfusion chamber. Results. Close intra-arterial infusion to the duodenal segment of 5-HT (20–200 nmol kg−1 h−1) dose-dependently increased duodenal mucosal HCO3 secretion. A higher dose (2000 nmol kg−1 h−1) did not further increase secretion. Responses were inhibited by the ganglionic blocker and nicotinic receptor antagonist hexamethonium, and were abolished by the 5-HT4 receptor antagonist SB 204070. The 5-HT3 antagonist tropisetron, in contrast, caused only slight inhibition. Viable human and rat duodenal enterocytes responded to 5-HT (100–500 nM) with an increase in intracellular calcium concentration. Pretreatment with SB 204070 or removal of external calcium abolished the response. Conclusions. Stimulation of the duodenal protective secretion by 5-HT thus involves receptors of the 5-HT4 subtype as well as nicotinic transmission, the myenteric plexus being a likely location. In addition, serotonin acts on enterocyte membrane receptors, inducing intracellular calcium signalling.

Estrogen induces phospholipase A 2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells

The principal secreted estrogen, 17β-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17β-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2–3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A 2 (PLA 2) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA 2α to perinuclear membranes occurred 1–2 min after 17β-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA 2α at serine residue 505. The phosphorylation and translocation of cPLA 2 were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA 2 could be stimulated by membrane impermeable 17β-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA 2 and the eicosanoid biosynthetic pathway are involved in the 17β-estradiol induced rapid calcium responses of breast cancer cells.

Plasma membrane phosphoinositide organization by protein electrostatics

Phosphatidylinositol 4,5-bisphosphate (PIP2), which comprises only about 1% of the phospholipids in the cytoplasmic leaflet of the plasma membrane, is the source of three second messengers, activates many ion channels and enzymes, is involved in both endocytosis and exocytosis, anchors proteins to the membrane through several structured domains and has other roles. How can a single lipid in a fluid bilayer regulate so many distinct physiological processes? Spatial organization might be the key to this. Recent studies suggest that membrane proteins concentrate PIP2 and, in response to local increases in intracellular calcium concentration, release it to interact with other biologically important molecules.

Transition of Human Embryonic Stem Cell-Derived Endothelial Cells to Smooth Muscle Cells in Culture as a Model for Vascular Development.

There is now growing evidence that embryonic stem cells provide an important resource to define the cellular and molecular mechanism of vascular development, and may serve as a potential source of cells for vascular repair. In our study, human ES cells (hESCs), H9 cell line, were differentiated by stromal cell co-culture with mouse bone marrow-derived stromal cell line S17 for 10–15 days. After this time, flow cytometry analysis confirmed the presence of a cell population with expression of surface antigens typical of endothelial cells (ECs). The hESC-derived cells were sorted for specific subpopulations of CD34+, CD31+, Flk1+ and Tie2+ cells using immunomagnetic selection to enrich for endothelial precursors. These sorted cells were cultured on fibronectin-coated plates in EGM2 media. Under these conditions, the cells assume EC morphology. The putative hESC-derived ECs expressed several EC markers including Flk1, Tie2, CD143, CD146, and bound to the lectin UEA-1. Gene expression analysis by RT-PCR further confirmed expression of transcripts for endothelial genes: Flk1, CD31, CD34, Tie2, eNOs, vWF and VE-Cadherin. Furthermore, the ECs were functional as shown by their ability to take up acLDL and form capillary-like structures when replated on Matrigel. To evaluate the smooth muscle cell (SMC) potential of this hESC-derived EC population, culture conditions were changed to media containing FBS, TGF β and PDGF-BB. Under these SMC-conditions, the cell populations converted to a flatter morphology and acquired intracellular fibrils. These cells expressed smooth muscle specific markers, as determined by immunohistochemistry: α-SMC actin, calponin and SM22. Q-RT-PCR confirmed a remarkable increase in expression of transcripts specific for SMC: α-SMC actin, calponin, SM22, smoothelin, myocardin. Importantly, we also found concomitant increased expression of 2 genes APEG-1and CRP2/SmLIM, preferentially expressed in arterial SMCs. At the time when SMC-gene expression increased, there was a corresponding dramatic decrease in expression of transcripts for endothelial genes. Notably, HUVEC cells treated with the same SMC-conditions did not develop into SMCs, suggesting this transition potential is unique to hESC-derived cells. Next, we used two functional tests to further evaluate the hESC-derived SMCs. First, we examined increase in intracellular calcium concentration evoked by 9 different agonists. The majority of the SMC population responded to bradykinin, oxytocin, endothelin-1, histamine and ATP, with fewer cells demonstrating a response to serotonin, vasopressin, norephinephrine and carbachol, consistent with a smooth muscle phenotype. In contrast, the hES-derived ECs responded to endothelin-1, histamine, bradykinin, as well as carbachol, with little response to oxytocin or the other agonists. Finally, we demonstrate that co-culture of hESC-derived SMCs together with hESC-derived ECs form ordered vascular structures composed of both cell types when cultured in a 3-dimensional Matrigel. These studies demonstrate that populations of hESC-derived ECs can convert to SMCs based on defined culture conditions. Further studies are now needed to identify whether this transition is the result of bipotential progenitor cells; or, if specific differentiated cells switch between these lineages. Alternatively, differentiated hESCs may produce progenitor cells specific for each lineage that are retained within the EC population.

Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood. Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular events associated with SVA suppression of BSA’s activity. In this communication, we demonstrate that SVA suppresses the BSA-induced increase of intracellular calcium concentration ([Ca 2+] i), intracellular pH (pH i), the cAMP level, PKA activity, protein tyrosine phosphorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).

Nonpathogenic, Environmental Fungi Induce Activation and Degranulation of Human Eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.

Newborn lamb coronary artery reactivity is programmed by early gestation dexamethasone before the onset of systemic hypertension

Exposure of the early gestation ovine fetus to exogenous glucocorticoids induces organ-specific alterations in postnatal cardiovascular physiology. To determine whether early gestation corticosteroid exposure alters coronary reactivity before the development of systemic hypertension, dexamethasone (0.28 mg x kg(-1) x day(-1)) was administered to pregnant ewes by intravenous infusion over 48 h beginning at 27 days gestation (term, 145 days). Vascular responsiveness was assessed in endothelium-intact coronary arteries isolated from 1-wk-old steroid-exposed and age-matched control lambs (N = 6). Calcium imaging was performed in fura 2-loaded primary cultures of vascular smooth muscle cells (VSMC) from the harvested coronary arteries. Early gestation steroid exposure did not significantly alter mean arterial blood pressure or coronary reactivity to KCl, thromboxane A(2) mimetic U-46619, or ANG II. Steroid exposure significantly increased coronary artery vasoconstriction to acetylcholine and endothelin-1. Vasodilatation to adenosine, but not nitroprusside or forskolin, was significantly attenuated following early gestation steroid exposure. Endothelin-1 or U-46619 stimulation resulted in a comparable increase in intracellular calcium concentration ([Ca(2+)](i)) in coronary VSMC isolated from either dexamethasone-treated or control animals. However, the ANG II- or KCl-mediated increase in [Ca(2+)](i) in control VSMC was significantly attenuated in VSMC harvested from dexamethasone-treated lambs. Coronary expression of muscle voltage-gated l-type calcium channel alpha-1 subunit protein was not significantly altered by steroid exposure, whereas endothelial nitric oxide synthase expression was attenuated. These findings demonstrate that early gestation glucocorticoid exposure elicits primary alterations in coronary responsiveness before the development of systemic hypertension. Glucocorticoid-induced alterations in coronary physiology may provide a mechanistic link between an adverse intrauterine environment and later cardiovascular disease.

Non-specific effects of leukotriene synthesis inhibitors on HeLa cell physiology

We examined the effects of various leukotriene synthesis inhibitors on calcium signalling in HeLa cells, before and after transfection with BLT 1. All of the inhibitors studied were found to reduce increases in intracellular calcium concentration induced by BLT 1, but also by an ionophore or activation of various G-protein coupled receptors, regardless of BLT 1 expression. In order to explore the mechanism of these apparently general effects we examined HeLa cell expression of leukotriene receptors and biosynthetic enzymes and found that the genes for key leukotriene synthesis enzymes and all of the leukotriene receptors were not expressed. Leukotrienes are involved in the pathology of a variety of cancers, and for HeLa cells leukotrienes have been reported to be important for aspects of the carcinogenic phenotype. We find that leukotriene synthesis inhibitors have non-specific effects, so careful controls are necessary to avoid interpreting non-specific effects as evidence for leukotriene involvement.