Increase In Intracellular Calcium Levels Research Articles

Natriuretic peptide receptor-A is functionally expressed on bullfrog retinal Müller cells

By the patch clamp technique, whole-cell currents induced by brain natriuretic peptide (BNP) from isolated bullfrog retinal Müller cells were studied. Application of 100 nM BNP induced a sustained inward current from these cells with a reversal potential of about 0 mV, and the current could be completely blocked by anantin, an antagonist of the A-type NP receptor (NPR-A) and CdCl 2, a blocker of cyclic nucleotide-gated (CNG) non-selective cation channels. Likewise, perfusion with the membrane-permeable cGMP analog 8-bromoguanosine-3′,5′-cyclic monophosphate (8Br-cGMP) caused effects that are similar to those of BNP. Moreover, application of BNP failed to induce any current in the presence of 1 mM 8Br-cGMP. By calcium imaging, we further showed a significant increase in intracellular calcium levels ([Ca 2+] i ) of all parts of Müller cells, including the endfoot, soma and processes following the perfusion of BNP, and the increase could be blocked by anantin. All these results suggest that NPR-A is expressed in bullfrog Müller cells, and activation of the receptor causes an increase of intracellular cGMP levels that activates CNG channels and thereby results in an increased calcium influx.

Receptors in Spermatozoa: Are They Real?

Receptors in Spermatozoa: Are They Real?

S100A7 (Psoriasin): a Story of Mice and Men

S100 proteins are calcium-regulated proteins that regulate fundamental biological processes. S100A7 (psoriasin), functions as a transglutaminase substrate/cornified envelope precursor, signal transduction protein, chemokine, and antibacterial protein in normal epidermis. S100A7 is markedly increased in epidermal hyperproliferative disorders. The murine homolog of S100A7 and S10015 has been identified, providing a valuable tool for studying the regulation and function of this protein in epidermis.

전립선암세포에 있어서 폴리아민에 의한 칼슘신호와 세포사멸

폴리아민은 거의 모든 세포에 있어서 성장과 분화에 필수적인 물질이다. 이들 폴리아민의 기작은 상당히 복잡하고 다양하여 그 정확한 작용기전은 아직 확실하지가 않다. 본 논문에서는 폴리아민이 세포 증식을 유도 하기도 하지만 일정농도 이상에서는 오히려 세포사멸을 초래한다는 결과를 밝히고자 한다. 본 실험에 사용된 인간의 전립선 암세포(LNCaP cells)에 있어서, 폴리아민 가운데 putrescien은 세포 증식에 거의 영향을 미치지 않았으나 spermidine과 spermine의 경우 10 <TEX>${\mu}M$</TEX> 이하에서는 세포 증식을 촉진하였다. 그러나, 20 <TEX>${\mu}M$</TEX> 이상의 농도에서는 농도와 시간의존적으로 세포사별을 유도 하였다. 폴리아민 처리에 의한 세포사멸의 초기과정인 핵 응축과 염색질 condensation이 Hoechst와 PI 염색에서 뚜렷이 관찰되었다. 또한, 폴리아민 처리시 anti-apoptotic protein으로 알려진 Bcl-2 protein의 발현은 거의 완전히 억제된 반면, pro-apoptotic protein으로 알려진 Bax의 발현은 현저히 증대되었다. 본 연구의 결과에 따르면, 폴리아민에 의해서 유도되는 세포사멸은 세포 내 칼슘농도 변화에 의한 것으로 사료된다. 전립선 암세포에 있어서 폴리아민 처리시 시간과 농도 의존적으로 세포 내 칼슘농도가 증가되었다. 세포막을 통한 칼슘이동을 억제하는 nifedipine과 flufenamic acid 등의 억제제를 처리한 실험 결과 세포내 칼슘증가는 세포 내부의 저장소로부터 칼슘의 유출보다는 주로 세포막상에 있는 비선택적 칼슘통로를 통한 외부의 칼슘 유입에 의한 것으로 판단된다. Polyamines are essential for the normal cell growth and differentiation. They are also known to have paradoxical dual effects on cell proliferation. In this paper we show that the excess amount of polyamines induces apoptosis through the modulation of calcium signaling in LNCaP human prostate cancer cells. Polyamines, particularly spermidine and spermine, stimulated cell proliferation at a lower concentration (under 10 <TEX>${\mu}M$</TEX>), but it inhibited cell viability at a higher concentration (40 <TEX>${\mu}M$</TEX>). The levels of intracellular <TEX>$Ca^{2+}$</TEX> concentration were increased only at a high concentration of polyamines treatment without any noticeable changes at lower concentrations. Nifedipine did not alter the increase of polyamine-induced <TEX>$Ca^{2+}$</TEX> levels, but flufenamic acid totally abolished the increase of intracellular <TEX>$Ca^{2+}$</TEX> levels. These results mean that polyamines induce <TEX>$Ca^{2+}$</TEX> influx from the surroundings through nonselective cation channels on the cell membrane. The expression of Bcl-2 protein was almost completely blocked, but the level of Bax protein was increased dramatically in the cells treated with high concentration of polyamine. The present study shows that polyamines at a high concentration induce apoptosis through the modulation of intracellular calcium signaling. The increase of intracellular calcium level induced by polyamines, was possibly a result from the extracellular calcium influx through the nonselective cation channels.

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.

Synaptotagmin I is involved in the regulation of cortical granule exocytosis in the sea urchin

Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis.

Transcriptional basis of lymphocyte tolerance

Signaling through lymphocyte antigen receptors has the potential to initiate several distinct outcomes: proliferation, differentiation, apoptosis, or functional unresponsiveness. Expansion and differentiation of effector T cells is required for defense against foreign antigens, whereas functional unresponsiveness, termed anergy, is a cell-intrinsic mechanism that contributes to peripheral self-tolerance. Other mechanisms of peripheral tolerance include the 'dominant' tolerance imposed by regulatory T cells and immunosuppression mediated by interleukin-10 and transforming growth factor-beta. T- and B-cell antigen receptor ligation induces an increase in intracellular calcium levels as well as activating additional signaling pathways that are further potentiated by costimulatory receptors. In this review, we argue that cell-intrinsic programs of peripheral anergy and tolerance are imposed by sustained calcium signaling in lymphocytes. We address in particular the role of the calcium-dependent transcription factor nuclear factor for activation of T cells, which is activated by antigen receptor stimulation and, depending on the presence or absence of input from its transcriptional partner, activator protein-1, dictates two distinct transcriptional programs: activation or tolerance.

Monitoring platelet inhibition after clopidogrel with the Verify Now-P2Y12® rapid analyzer: The VERIfy Thrombosis risk ASsessment (VERITAS) study

Introduction Clopidogrel inhibits platelet P2Y12 ADP receptors, while ADP, as an inductor of aggregation, stimulates both P2Y12 and P2Y1 platelet receptors. Despite a clinical loading dose routine with clopidogrel, some patients still experience coronary stent thrombosis suggesting persistent platelet activation. The Verify Now-P2Y12 is a rapid assay that test platelet activity over 3 min and uses of the combination of ADP and prostaglandin E1 (PGE1) to directly measure the effects of clopidogrel on the P2Y12 receptor. ADP is used to maximally activate the platelets by binding to the P2Y1 and P2Y12 platelet receptors, while PGE1 is used to suppress the ADP-induced P2Y1-mediated increase in intracellular calcium levels. Objective The VERIfy Thrombosis risk ASsessment (VERITAS) was a prospective study designed to measure platelet response to clopidogrel therapy in subjects with multiple risk factors or history of vascular disease using this novel point-of-care assay. Methods 166 participants were enrolled in 4 participating sites. Data from 147 participants were analyzed after exclusion of 19 patients due to protocol violations. Platelets were assessed twice at baseline (before clopidogrel) and at 24 h post-loading 450 mg (110 participants) or 7 days after chronic clopidogrel treatment (75 mg/day) (37 patients). All participants received aspirin 81–325 mg for at least 2 days before the study enrollment. Results from the Verify Now-P2Y12 assay are reported in P2Y12 reaction units (PRU). Results Clopidogrel therapy resulted in a mean 64.0 ± 25.3% PRU reduction. No participant reached PRU inhibition below 10% of baseline. Distribution of PRU values for the Verify Now-P2Y12 assay shows a separation from baseline to post-clopidogrel assay values with some overlap due to high inter-individual variations in response. Conclusions Verify Now-P2Y12 is a reliable, fast and sensitive device suitable for monitoring of platelet inhibition during clopidogrel therapy.

Excitotoxic neuronal injury in acute homocysteine neurotoxicity: Role of calcium and mitochondrial alterations

In this study we tested if calcium imbalance and mitochondrial dysfunction, which have been implicated in the conventional mechanisms of excitotoxicity induced by glutamate (Glu), are also involved in homocysteine (Hcy) neurotoxicity. Primary cultures of rat cerebellar granule cells were incubated for 30 min in the presence of 25 mM d, l-Hcy or 1 mM Glu. At these concentrations both amino acids induced comparable neurodegeneration and chromatin condensation, evaluated after 24 h using the propidium iodide and Hoechst 33258 staining. These effects were partially prevented by cyclosporin A (CsA), but not FK506. Hcy-induced release of [ 3H]inositol phosphates and increase in intracellular calcium level (evaluated with fluo-3 fluorescent probe) were weakly expressed. Hcy- and Glu-induced mitochondrial swelling was visualized under electron microscope, and the release of Cytochrome c was evaluated using immunocytochemical method and confocal microscopy. Comparing to Glu, the effects of Hcy were slightly less expressed and less sensitive to CsA, while FK506 did not modify mitochondrial alterations. These data indicate that mitochondrial alterations play a similar role in acute Hcy and Glu neurotoxicity, although the mechanisms triggering Glu- and Hcy-evoked mitochondrial dysfunction seem to differ, Hcy toxicity being less dependent on calcium.

Isolation and Cultivation of Human Testicular Peritubular Cells: A New Model for the Investigation of Fibrotic Processes in the Human Testis and Male Infertility

Fibrotic remodeling, especially of the tubule wall, in testes of infertile men is common, but reasons or consequences of these striking changes are not known. Based on cell culture and ex vivo studies, we previously suggested that mast cells via their products tryptase and histamine are involved in the development of fibrosis. However, studies in a relevant human testicular model are required to further test this hypothesis and the mechanisms of testicular fibrosis in general. The objective of the study was the isolation, culture, and characterization of adult human testicular peritubular cells. Peritubular cells were obtained from biopsies of men suffering from obstructive azoospermia (n = 8) and varicocele (n = 2) but displaying normal spermatogenesis. Explant cultures were obtained from all biopsies. Immunostaining of the cultured cells and corresponding paraffin-embedded tissues with antibodies against markers of fibroblasts (CD90/Thy-1) and smooth muscle cells (alpha-smooth muscle actin) clearly proved their origin from the peritubular region. These cells displayed morphological features of myofibroblasts, and gene array analyses as well as immunohistochemistry revealed the predominant expression of extracellular matrix genes and genes coding for basement membrane components. The cultured cells retain receptors for the major mast cell products histamine and tryptase. The addition of histamine (100 microm) and the tryptase agonist peptide SLIGKV (10 microm) resulted in a transient increase in intracellular calcium levels, confirming the functionality of the receptors. We conclude that human peritubular cells are a novel model for the investigation of paracrine, including mast cell initiated, interactions in the human testis, which will allow the study of fibrotic processes underlying male idiopathic infertility.

A role for adrenomedullin in the pathogenesis of alveolar edema

Christ-Crain and colleagues [1] established in their work a close relationship between pro-adrenomedullin levels and severity of community acquired pneumonia. This raises the question of whether this peptide or its precursor adrenomedullin are involved in the pathogenesis of the respiratory compromise in pneumonia. Adrenomedullin induces nitric oxide (NO) production in endothelial cells through an increase in intracellular calcium levels, which activates NO synthetase [2]. NO has been linked to endotoxin induced acute lung injury in which the NO scavenger N-acetyl cysteine reduced exhaled NO levels and lung water [3]. The mechanism by which NO reduces alveolar fluid clearance and contributes to the extent of pulmonary fluid accumulation in lung injury has been clarified: NO reduces both the activity of apical alveolar epithelial sodium channels and the baso-lateral alveolar epithelial sodium-potassium ATPase, which regulate alveolar sodium and water absorption. Sodium absorption hereby generates the osmotic gradient, drawing alveolar fluid through alveolar epithelial aquaporin channels and paracellular pathways [4,5]. Future research needs to investigate the role of adrenomedullin in the generation of pulmonary NO production in lung injury and whether treatments leading to a reduction of adrenomedullin levels can reduce the severity and extent of alveolar edema in pneumonia and septicemia.

Macrophage migration inhibitory factor induces cell death and decreases neuronal nitric oxide expression in spinal cord neurons

Macrophage migration inhibitory factor is a potent proinflammatory cytokine; however, its role in spinal cord injury is poorly understood. Therefore, the aim of the present study was to investigate the effects of macrophage migration inhibitory factor on spinal cord neuron survival and viability. Due to the importance of nitric oxide metabolism in these events, part of our study was also focused on the influence of recombinant macrophage migration inhibitory factor on neuronal nitric oxide expression. Exposure of cultured mouse spinal cord neurons to macrophage migration inhibitory factor markedly increased cellular oxidative stress as measured by 2′,7′-dichlorofluorescein fluorescence and intracellular calcium levels. In addition, an antagonist of the inositol 1,4,5-triphosphate receptor, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, completely blocked the macrophage migration inhibitory factor-induced increase in intracellular calcium levels. Macrophage migration inhibitory factor treatment also decreased cell viability, increased cellular lactate dehydrogenase release, and induced chromatin condensation and aggregation in cultured spinal cord neurons. Finally, exposure to macrophage migration inhibitory factor markedly decreased expression and activity of neuronal nitric oxide, accompanied by a decrease in cellular guanosine 3′5′-cyclic monophosphate levels. The present results indicate that macrophage migration inhibitory factor can induce dysfunction of spinal cord neurons, leading to cell death through oxidative stress and intracellular calcium-dependent pathways.

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.

Vampire Bat Salivary Plasminogen Activator (Desmoteplase) Inhibits Tissue-Type Plasminogen Activator-Induced Potentiation of Excitotoxic Injury

In contrast to tissue-type plasminogen activator (tPA), vampire bat (Desmodus rotundus) salivary plasminogen activator (desmoteplase [DSPA]) does not promote excitotoxic injury when injected directly into the brain. We have compared the excitotoxic effects of intravenously delivered tPA and DSPA and determined whether DSPA can antagonize the neurotoxic and calcium enhancing effects of tPA. The brain striatal region of wild-type c57 Black 6 mice was stereotaxically injected with N-methyl-d-Aspartate (NMDA); 24 hour later, mice received an intravenous injection of tPA or DSPA (10 mg/kg) and lesion size was assessed after 24 hours. Cell death and calcium mobilization studies were performed using cultures of primary murine cortical neurons. NMDA-mediated injury was increased after intravenous administration of tPA, whereas no additional toxicity was seen after administration of DSPA. Unlike DSPA, tPA enhanced NMDA-induced cell death and the NMDA-mediated increase in intracellular calcium levels in vitro. Moreover, the enhancing effects of tPA were blocked by DSPA. Intravenous administration of tPA promotes excitotoxic injury, raising the possibility that leakage of tPA from the vasculature into the parenchyma contributes to brain damage. The lack of such toxicity by DSPA further encourages its use as a thrombolytic agent in the treatment of ischemic stroke.

Vimentin-Dependent Spatial Translocation of an Activated MAP Kinase in Injured Nerve

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

AT receptors and L-type calcium channels: functional coupling in supersensitivity to angiotensin II in diabetic rats

The study was designed to investigate the role of calcium channels in enhanced angiotensin II (Ang II)-induced contraction in thoracic aortic rings from diabetic rats. Ang II-induced isometric tension was studied in thoracic aortic rings isolated from control or streptozotocin-induced (8 weeks) diabetic rats. Saturation binding studies at AT1 receptors and L-type calcium channels were performed using [3H] Ang II and [3H] PN200110, respectively. Ang II-induced calcium influx was studied in fura-2-loaded single vascular smooth muscle cells isolated from thoracic aorta of control and diabetic rats. Ang II did not induce contraction in calcium-free Krebs. In the presence of extracellular calcium, increased Emax (mg/mm2) and pD2 to Ang II was observed in aortic rings from diabetic (795.54+/-38.19; 8.27+/-0.12) compared to control (230.09+/-25.45; 7.68+/-0.22) rats, respectively. Nimodipine but not verapamil or diltiazem dose-dependently blocked the Ang II-induced contractions in a noncompetitive manner and its -log IC50 was significantly lower in aortic rings from diabetic (8.81+/-0.10) compared to control (9.34+/-0.11) rats. The Ang II-induced increase in intracellular calcium levels was significantly enhanced (2.5-fold) in vascular smooth muscle cells from diabetic rats. AT1 receptor saturation binding with [3H] Ang II revealed a significantly higher affinity (nM) and Bmax (pmol/mg protein) in aortic vascular membrane preparation from diabetic (0.31+/-0.04; 64.18+/-2.4) compared to control (0.52+/-0.02; 47.81+/-2.8) rats, respectively, while L-type calcium channel saturation binding with [3H] PN200110 showed a higher affinity (nM) with no change in the Bmax (fmol/mg protein) in diabetic (0.74+/-0.08; 4.52+/-0.40) compared to control (1.49+/-0.32; 5.43+/-0.60) aortic membranes, respectively. Our results suggest that Ang II-induced contraction is dependent on extracellular calcium, and enhanced functional coupling of AT1 receptors and DHP-sensitive L-type calcium channels results in supersensitivity to Ang II in thoracic aorta isolated from diabetic rats.

Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus

In several neurological disorders including hyperhomocysteinemia, homocysteine (Hcy) accumulates in the brain, and acts as a potent neurotoxin. However, the molecular mechanisms induced by increased levels of Hcy in brain are not well understood. Here we show an activation of the extracellular signal-regulated kinases (ERK1 and ERK2) and the downstream nuclear targets Elk-1 and calcium/cAMP response element binding protein, in the hippocampus of cystathionine beta synthase deficient mice, a murine model of hyperhomocysteinemia. An ex vivo model of hippocampal slices allowed us to reproduce Hcy -induced ERK activation and to unravel the mechanisms responsible of this activation. Of interest, N-methyl- d-aspartate (NMDA), non-NMDA and metabotropic glutamate receptor antagonists all blocked Hcy -induced ERK activation. Moreover, the ERK activation was blocked in the presence of Na +-channel blocker tetrodotoxin, indicating the existence of a trans-synaptic activity in ERK activation by Hcy in hippocampal slices. The effects of Hcy on ERK cascade activation were also dependent on calcium influx, CaMK-II, PKC as well as PKA activation. Thus, altogether these data support a role of Hcy on ERK activation, via complex mechanisms, starting with a control of glutamate release, which in turn activates ionotropic and metabotropic receptor subtypes and produces increases in intracellular calcium levels.

280 EFFECTS OF β-ENDORPHIN AND NALOXONE ON INTRACELLULAR CALCIUM LEVELS IN CUMULUS CELLS OF EQUINE OOCYTES

Changes in intracellular calcium levels in the cumulus oocyte complex (COC) have a crucial role in oocyte maturation. In previous studies we demonstrated that the μ-opioid receptor is expressed in the bovine COC and participates in the signaling associated with oocyte maturation, by inducing an intracellular calcium increase (Dell'Aquila ME et al. 2002 Mol. Reprod. Dev. 63, 210–222). In this work we evaluated modifications of intracellular calcium induced by β-endorphin (β-end) or Naloxone (Nx) in cumulus cells of equine oocytes in relation to the time of the year and cumulus morphology at retrieval. Cumulus cells, isolated by mechanical treatment from compact (Cp, n = 120) or expanded (Exp, n = 120) COCs, recovered from the ovaries of slaughtered mares (follicles &lt;20 mm in diameter) during anestrus, breeding season, spring transition, and autumnal transition, were cultured for 24 h and loaded with 5 μM Fura2-AM for microspectrofluorometric measurements of cytoplasmic ionized calcium (Dell'Aquila et al., 2002). The changes in β-end (30 μM)- or Nx (1mM and 10 μM)-induced calcium concentration were calculated in single cells (n = 194) and are expressed as Δ fluorescence (Fmaximal effect – Fbaseline) before and after 1-min perfusion with the drugs. The use of 1 mM Nx induced a significant increase of intracellular calcium levels in cumulus cells of oocytes recovered in all periods of the year in both Cp and Exp (P &lt; 0.01). The addition of 10 μM Nx or 30 μM β-end significantly increased intracellular calcium only in cumulus cells from oocytes recovered in anestrus (P &lt; 0.05). These results confirm previous observations, carried out on bovine oocytes, in which Nx behaved as a μ-receptor agonist when used at high concentration (Dell'Aquila et al. 2002). The effects of β-end and Nx may be explained in terms of a binding of the two subtances at the μ-receptor with consequent intracellular calcium increases due to extracellular calcium entry or depletion of intracellular stores. These findings could be related to differential espression and/or activation status of the μ-opioid receptor in COCs retrieved in different seasons. These substances can be used to modulate intracellular calcium in the equine COCs, and consequent effects on the stimulation/inhibition of oocyte maturation in this species need to be further investigated. This work was supported by Grant MIUR COFIN PRIN 2003.

Delayed and Sustained Activation of Extracellular Signal-regulated Kinase in Human Keratinocytes by UVA: IMPLICATIONS IN CARCINOGENESIS

Exposure to the sun's UV radiation appears to be the most important environmental factor involved in the development of skin cancer. UVA is the major portion of UV radiation in sunlight and is considered to be a human carcinogen. In this study, we have investigated the delayed and sustained activation of ERK MAPK by UVA exposure. In parallel, a delayed Ras activation with a similar time course was observed after UVA exposure. The activated Ras was found to be localized in endomembranes such as the Golgi apparatus instead of plasma membranes. Expression of dominant negative Ras (N17Ras) abolished ERK activation by UVA. The presence of AG1478, an epidermal growth factor (EGF) receptor (EGFR) kinase inhibitor, had no effect on ERK or Ras activation, indicating that EGFR kinase activity is not involved in ERK activation by UVA. In contrast, protein kinase C (PKC) depletion by chronic 12-O-tetradecanoylphorbol-13-acetate treatment nearly abolished UVA-induced ERK and Ras activation. The presence of the Ca(2+)-dependent-PKC inhibitor Go6976 had a similar effect. These findings suggest that ERK activation by UVA is mediated by PKC in a Ras-dependent pathway. In addition, a gradual increase in intracellular calcium level after UVA exposure was detected by flow cytometry. The presence of the PLC inhibitor U73122 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) blocked both ERK and Ras activation, suggesting that both PLC and calcium are required for ERK activation. Our findings demonstrated that, different from UVC and UVB, UVA-induced delayed and sustained ERK activation is EGFR kinase activity-independent, but PLC/calcium/PKC-mediated. The delayed and sustained ERK activation provides a survival signal to human HaCaT keratinocytes, which may serve as an important mechanism for cell transformation and potential skin carcinogenesis in vivo caused by UVA exposure.

Nitric oxide donors induce calcium-mobilisation from internal stores but do not stimulate catecholamine secretion by bovine chromaffin cells in resting conditions

The potential role of nitric oxide (NO) donors and peroxynitrites on both basal catecholamine (CA) secretion and modulation of calcium levels has been investigated in primary cultures of bovine chromaffin cells. NO donors did not modulate catecholamine secretion, while peroxynitrites induced a time dose-dependent increase in basal CA secretion. Two facts may explain the lack of these compounds on basal CA secretion. NO donors induce, on the one hand, an increase in intracellular calcium levels by depletion of internal IP 3-stores from endoplasmic reticulum. On the other hand, a small calcium influx through N-type voltage-dependent calcium channels (VDCC), which seem not to be coupled to exocytosis of adrenaline and noradrenaline in chromaffin cells. Both effects, calcium-mobilisation from internal stores and calcium entry through N-type VDCC are mediated by cGMP synthesis. In contrast, peroxynitrites induce an increase in basal CA secretion by both a decrease of intracellular catecholamine content and a toxic effect on cellular membrane. All these results, taken together, could explain contradictory results in the literature on the role of NO on basal catecholamine secretion and on modulation of intracellular calcium in chromaffin cells.