Cytosolic Calcium Ion Research Articles

Calcium spiking activity and baseline calcium levels in ROS 17/2.8 cells exposed to extremely low frequency electromagnetic fields (ELF EMF)

Purpose : To determine whether extremely low frequency electromagnetic fields can alter average free cytosolic calcium ion concentrations [Ca 2+ ] i and transient increases in [Ca 2+ ] i in populations of ROS 17/2.8 cells. Materials and methods : Cells loaded with the calcium-selective luminescent photoprotein, aequorin, were placed in the bottom of a sample chamber, which was inserted into the gap of a previously described air gap reactor system where they were exposed either to sinusoidal magnetic fields at a variety of frequencies and flux densities or to sham conditions. Real-time recordings of photon counts due to aequorin luminescence were obtained and data were analysed with the use of probit plots. Results : Probit plots of data obtained from cells exposed to the various magnetic fields were virtually superimposable over the data obtained for the same cultures during pre- and post-exposure sham or no-field periods. Conclusion : These experiments provided no evidence for any effects of ELF EMF, either positive or negative, on either average [Ca 2+ ] i or on transient increases in [Ca 2+ ] i.

Increased inflammation and intracellular calcium caused by ultrafine carbon black is independent of transition metals or other soluble components

OBJECTIVESParticulate air pollution has been shown to cause adverse health effects, and the ultrafine particle component has been implicated. The aim of the present study was to investigate whether an...

Cholinergic stimulation enhances cytosolic calcium ion accumulation in mouse hippocampal CA1 pyramidal neurones during short action potential trains.

Acetylcholine is a regulatory cofactor for numerous activity-dependent processes of central nervous system development and plasticity in which increases in cytosolic calcium ion concentration ([Ca(2+)](cyto) couple membrane excitation to cellular changes. We examined how cholinergic receptor activation affects temporal and spatial aspects of increases in [Ca(2+)](cyto) during short trains of action potentials in hippocampal CA1 pyramidal neurones. Membrane-impermeant Ca(2+)-sensitive dye was introduced into the cytosol during whole-cell recordings, and Ca(2+)-dependent fluorescence was recorded from somatic, nuclear and proximal dendrite regions with high temporal resolution. In all neuronal compartments, the cholinergic agonist carbachol (5 microM) increased resting [Ca(2+)](cyto) and the maximum [Ca(2+)](cyto) attained during a short action potential train. Carbachol also slowed the recovery of [Ca(2+)](cyto) towards resting levels. The largest increases in peak cytosolic Ca(2+) concentration (delta [Ca(2+)](cyto) were seen in the dendrite and apical cell body, while relaxations of the carbachol-induced increase in delta [Ca(2+)](cyto) showed greater prolongation in the nucleus and basal cell body. Most significantly, the difference between Ca(2+) signals recorded before and during exposure to carbachol consistently showed a monotonic rise and smooth fall in all cell compartments, suggesting that the increase in [Ca(2+)](cyto) associated with each action potential was not altered by carbachol. Consistent with this view, changes in Ca(2+) signalling were not accompanied by changes in action potential waveforms. The effects of carbachol were partially reversed by simultaneous exposure to atropine, or partially inhibited by inclusion of heparin in the intracellular solution, indicating the involvement of muscarinic acetylcholine receptors and InsP(3)-sensitive Ca(2+)-release channels. Our data indicate that carbachol-induced slowing of [Ca(2+)]cyto relaxations after each action potential results in enhanced accumulation of Ca(2+) in the cytosol in the absence of changes in action potential-driven Ca(2+) entry. By modulating the time course of Ca(2+) signals, cholinergic stimulation may regulate the activation of Ca(2+)-dependent intracellular processes dependent on patterns of [Ca(2+)](cyto) changes.

Ruled by waves? Intracellular and intercellular calcium signalling.

The field of calcium signalling has evolved rapidly the last 20 years. Physiologists had worked with cytosolic Ca2+ as the coupler of excitation and contraction of muscles and as a secretory signal in exocrine glands and in the synapses of the brain for several decades before the discovery of cellular calcium as a second messenger. Development of powerful techniques for measuring the concentration of cytosolic free calcium ions in cell suspensions and later in single cells and even in different cellular compartments, has resulted in an upsurge in the knowledge of the cellular machinery involved in intracellular calcium signalling. However, the focus on intracellular mechanisms might have led this field of study away from physiology. During the last few years there is an increasing evidence for an important role of calcium also as an intercellular signal. Via gap junctions calcium is able to co-ordinate cell populations and even organs like the liver. Here we will give an overview of the general mechanisms of intracellular calcium signalling, and then review the recent data on intercellular calcium signals. A functional coupling of cells in different tissues and organs by the way of calcium might be an important mechanism for controlling and synchronizing physiological responses

Calcium dynamics and buffering in oculomotor neurones from mouse that are particularly resistant during amyotrophic lateral sclerosis (ALS)-related motoneurone disease.

Motoneurones are particularly vulnerable both in human forms of amyotrophic lateral sclerosis (ALS) and corresponding animal models of the disease. While most motoneurone populations are selectively impaired, oculomotor neurones are essentially resistant to ALS-related damage. Motoneurone vulnerability has been closely linked to disruptions of calcium signalling. To investigate underlying events, we performed a quantitative analysis of calcium homeostasis in oculomotor neurones from mice by simultaneous patch-clamp recordings in sliced tissue and microfluorometric-calcium measurements. Somatic calcium dynamics were investigated by using a computer-controlled microfluorometric system. In oculomotor neurones, basal calcium concentrations were around 80 nM and depolarisation-induced calcium responses were observed for membrane voltages positive to -40 u1u1u approximately mV1 approximately . Endogenous calcium homeostasis was quantified by using the 'added buffer' approach. The recovery phase of depolarisation-induced calcium transients was well approximated by a mono-exponential function with a decay time constant that showed a linear dependence on dye concentration. The extrapolated time constant in the absence of indicator dye was 1.7 +/- 0.2 s (n = 11 cells, 21C). Endogenous calcium binding ratios (kappa(s)) were found to be 264 +/- 25 (n = 11 cells), indicating that 99.6 % of cytosolic calcium ions were taken up by endogenous buffers. Recovery of calcium transients was characterised by an 'effective' extrusion rate gamma = 156 +/- 20 s-1 (n = 11 cells, 21 C). Endogenous calcium binding ratios in oculomotor neurones were 5- to 6-fold larger compared with those of more vulnerable motoneurones in the nucleus hypoglossus and spinal cord. In a first order approximation, they reduced the volume of local calcium elevations around open calcium channels, lowered peak amplitudes of global calcium transients for a given influx and prolonged calcium recovery times for a given set of uptake and extrusion mechanisms. With respect to motoneurone degeneration, our measurements suggest that the exceptional stability of oculomotor neurones partially results from a specialised calcium homeostasis based on high buffering capacities. Furthermore, they indicate that cellular adaptations that account for rapid calcium signalling in hypoglossal and spinal motoneurones enhance their vulnerability during ALS-related motoneurone disease.

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices.

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.

Power-frequency electromagnetic fields and the capacitative calcium entry system in SV40-transformed Swiss 3T3 cells.

The present study was designed to test the hypothesis that a 60 Hz electromagnetic field could affect the influx of calcium ions across the plasma membrane through the so-called capacitative calcium entry system. Recordings of cytosolic calcium-ion concentrations in SV40-transformed Swiss 3T3 cells were obtained in real time during exposure to magnetic fields ranging from 0.3-50 mT or to sham conditions using the calcium-sensitive photoprotein aequorin. This was done for cell populations whose capacitative entry system was activated by either bradykinin or thapsigargin under a variety of experimental conditions. No effects of the magnetic field were observed on bradykinin-induced calcium transients and, with the exception of a small but statistically significant increase observed in experiments performed at 50 mT, no effects of the fields were observed on baseline calcium levels prior to or after such transients. The magnetic fields also had no effects on the size or kinetics of any of the thapsigargin-induced calcium transients. Overall, the data fail to support the hypothesis tested in this work.

The Ca2+-transporting activity of rat liver microsomes in response to protein undernutrition: implications for liver tumor promotion.

The effect of protein undernutrition on the activity of the smooth endoplasmic reticulum (microsomal) Ca2+-ATPase (SERCA) was investigated. After 12 weeks of ad libitum ingestion of low protein diet (5% protein), a significant depression (p <0.05) of liver ER Ca2+-ATPase activity (68.6% depression) was observed. However, no significant effects on cytochrome P450 activity and relative liver weight were found. It is proposed that low protein diet by inhibiting the rat liver SERCA activity, might increase the cytosolic free calcium ion concentration ([Ca2+]) and promote the development of liver tumor. The possible mechanisms of low protein diet induced inhibition of SERCA activity are highlighted.

Procoagulant Expression in Platelets and Defects Leading to Clinical Disorders

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant microvesicles have been demonstrated in several clinical conditions, such as thrombotic and idiopathic thrombocytopenia, disseminated intravascular coagulation, and HIV-1 infection, and have been found to be associated with fibrin in thrombosis. Procoagulant microvesicles may also be formed from other cells as a result of apoptosis.

Role of guanylyl cyclase in fertilisation of sea urchin eggs

A transient increase in cytosolic free calcium ion concentration ([Ca2+]i) (Ca2+-transient) takes place in the early stages of fertilisation of sea urchin eggs as well as in other animal eggs (Miyazakiet al., 1993). This transient increase in [Ca2+]ipropagates across the egg as a Ca2+wave, which is thought to be a necessary and sufficient event for egg activation (Whitaker &amp; Swarm, 1993). In sea urchin eggs, the rise in [Ca2+], is caused by release of Ca2+from the endoplasmic reticulum (ER) via one or both of two pathways: (a) inositol 1,4,5-trisphosphate (IP3) and the inositol 1,4,5-trisphosphate receptor/channel (IP3R) or (b) cADP-ribose (cADPR) and/or cGMP and the ryanodine receptor/channel (RyR) (Berridge, 1993). The signalling pathways from sperm to ER of eggs are not yet fully explained. Recent evidence from two lines of experiments has excited more controversy. First, intracellular injection of SH2 domain of phospholipase Cγ, which produced IP3, completely inhibited the increase in [Ca2+]i(Carrollet al., 1999). Another series of experiments showed that nitric oxide (NO) gas was produced in sperm during their acrosome reaction and in eggs during fertilisation, and that the intracellular injection of NO synthase caused egg activation (Epel, this supplement). NO gas is expected to stimulate the production of cGMP by activating soluble guanylyl cyclase (Garthewaite, 1991). Thus, it seems that direct measurements of the second messenger candidates during sea urchin fertilisation are essential to an understanding of the calcium signalling pathway. We previously measured the IP3, cGMP and cADPR contents of sea urchin eggs, and compared the time courses of their changes with that of the [Ca2+]ichange (Kurodaet al., 1997). We now examine further the involvement of guanylyl cyclase in the Ca2+signalling pathway at fertilisation of sea urchin eggs.

A study of the fluorescence measurement using a 96-well microplate by a remodelled parallel luminescent measuring system.

To develop an index to measure alveolar macrophage activity, the fluorescent technique for detection of calcium flux was paid special attention. In this study, a parallel luminescence measuring system was remodelled for fluorescence measurement using a 96-well microplate. The fluorescence indicators widely used to measure cytosolic free calcium ion concentration require excitation at ultraviolet (UV) wavelengths. Instruments to produce UV wavelengths are expensive compared to those used to produce visible wavelengths, and UV wavelengths are potentially injurious to cells. To avoid these problems, Fluo 3 (excitation wavelength in the visible range) was used as the fluorescent dye for detecting calcium ions. The parallel luminometer was remodelled successfully for fluorescent measurement as assessed by the results obtained from the measurements of a common fluorescent dye, fluorescein. Concentrations of free calcium ions were measured using Fluo 3 at 37 degrees C to consider the measurement of calcium flux in living cells. Although a linear relationship between concentrations of free calcium ions and fluorescence were observed, a diminution of fluorescence over time was also observed. To measure calcium flux in living cells, further instrumental and experimental improvements are thus needed.

Calcium dynamics and buffering in motoneurones of the mouse spinal cord.

1. A quantitative analysis of endogenous calcium homeostasis was performed on 65 motoneurones in slices of the lumbar spinal cord from 2- to 8-day-old mice by simultaneous patch-clamp and microfluorometric calcium measurements. 2. Somatic calcium concentrations were monitored with a temporal resolution in the millisecond time domain. Measurements were performed by using a monochromator for excitation and a photomultiplier detection system. 3. Somatic calcium signalling was investigated during defined voltage-clamp protocols. Calcium responses were observed for membrane depolarizations positive to -50 mV. A linear relation between depolarization time and free calcium concentrations ([Ca2+]i) indicated that voltage-dependent calcium influx dominated the response. 4. Endogenous calcium homeostasis was quantified by using the 'added buffer' approach. In the presence of fura-2 and mag-fura-5, calcium transients decayed according to a monoexponential function. Decay-time constants showed a linear dependence on dye concentration and the extrapolated constant in the absence of indicator dye was 371 +/- 120 ms (n = 13 cells, 21 C). 5. For moderate elevations (< 1 microM), recovery kinetics of depolarization-induced calcium transients were characterized by a calcium-independent, 'effective' extrusion rate gamma = 140 +/- 47 s-1 (n = 13 cells, 21 C). 6. The endogenous calcium binding ratio for fixed buffers in spinal motoneurones was kappaB' = 50 +/- 17 (n = 13 cells), indicating that less than 2 % of cytosolic calcium ions contributed to [Ca2+]i. 7. Endogenous binding ratios in spinal motoneurones were small compared to those found in hippocampal or cerebellar Purkinje neurones. From a functional perspective, they provided motoneurones with rapid dynamics of cytosolic [Ca2+]i for a given set of influx, extrusion and uptake mechanisms. 8. With respect to pathophysiological conditions, our measurements are in agreement with a model where the selective vulnerability of spinal motoneurones during excitotoxic conditions and motoneurone disease partially results from low endogenous calcium buffering.

Mechanisms underlying endothelin’s inhibition of FSH-stimulated progesterone production by ovarian granulosa cells

In previous studies in porcine granulosa cell cultures, endothelin-1 (ET-1) was shown to inhibit FSH-stimulated cAMP and progesterone accumulation, and to increase inositol phosphate formation and cytosolic calcium ion concentration. The latter results suggest an action of ET-1 via the activation of phospholipase C. Here we have investigated the following experimental questions. (1) Does ET-1 activate PKC in ovarian cells? (2) Does the cellular mechanism(s) whereby ET-1 interferes with the steroidogenic action of FSH in granulosa cells involve an impairment of cAMP generation or action? And (3) how does the site(s) of the inhibitory effect(s) of ET-1 and TPA on FSH-stimulated progesterone accumulation in cultured granulosa cells compare? In the present investigation, ET-1 (1 μM) induced rapid cytosol-to-membrane translocation of [ 3H]phorbol 12,13-dibutyrate binding sites, indicating protein kinase C (PKC) activation. At 24 or 48 h, ET-1 inhibited FSH-, but not forskolin (1 μM)-induced, cAMP accumulation. Cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) messenger RNA (mRNA) accumulation was stimulated by FSH, 8-bromo-cAMP (8Br-cAMP, 0.5 mM) and forskolin. ET-1 significantly inhibited this effect of FSH, but not the effects of 8Br-cAMP and forskolin. Progesterone production decreased commensurately with this inhibitory action of ET-1 on the FSH-stimulated accumulation P450scc mRNA. The PKC activator, 12- O-tetradecanoyl-phorbol-13-acetate (TPA), suppressed steroidogenesis stimulated by forskolin and 8Br-cAMP as well as FSH. In conclusion, ET-1 inhibited FSH-stimulated cAMP accumulation, P450scc expression, and progesterone production in porcine granulosa cell cultures. The data are compatible with pre-adenylate cyclase site of action. Although ET-1 activated PKC, TPA, unlike ET-1, seems to inhibit steroidogenesis by interfering with cAMP action.

Mechanisms of calcium transport in human endothelial cells subjected to hyperthermia

We have probed the mechanisms by which severe heat alters cytosolic calcium ion concentrations (Cai) in individual cultured human endothelial cells (ATCC ♯1998). Cells adhering to glass coverslips were heated to as high as 50°C and Cai determined by means of a fluorescence laser imaging system using the calcium-sensitive dye, indo-1, in the presence of thapsigargin, and in Na-free and Ca-free media. Baseline Cai varied between 175–225 nM. When cells were heated to 50°C in a complete Ca-containing medium, there was first a transient fall in Cai, then a rapid rise of 50–100 nM in Cai, followed by a slower, secondary rise of 50–75 nM. Depleting the intracellular calcium stores with thapsigargin blocked both the transient fall and the secondary rise in Cai. Placement of the cells into a Ca-free medium blocked both the transient fall and the initial rapid rise, while use of a Na-free buffer prevented the initial rapid rise only. These data suggest that in human endothelial cells, extreme heat accelerates the CaATPase pumps of the intracellular Ca stores causing the transient fall in Cai which is soon followed by activation of the reverse mode of the Na/Ca exchanger to cause the initial rapid rise in Cai. The Ca-release channels of the intracellular stores become activated by heat to cause the secondary, slow rise in Cai. This preliminary work indicates that the application of heat to cultured cells can be a useful probe to examine the kinetics and unmask mechanisms of intracellular Ca fluctuations.

Methanol Elevates Cytosolic Calcium Ions in Cultured Canine Cerebral Vascular Smooth Muscle Cells: Possible Relation to CNS Toxicity

LI, W., T. ZHENG, J. WANG, B. T. ALTURA AND B. M. ALTURA. Methanol elevates cytosolic calcium ions in cultured canine cerebral vascular smooth muscle cells: Possible relation to CNS toxicity. ALCOHOL 18(2/3) 221–224, 1999.—Acute exposure of cultured canine cerebral vascular smooth muscle cells to methanol (10–400 mM) results in concentration-dependent elevation of the concentration of intracellular free calcium ion ([Ca 2+] i) as measured with the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca 2+] i in the cerebral vascular smooth muscle cells was 89.3 ± 5.3 nM. Exposure of these cells to 10 mM methanol for only 5 min resulted in significant elevation in [Ca 2+] i (i.e., to 105.7 ± 4.6) ( p < 0.05). Methanol (10 mM) is a concentration found in the blood of victims demonstrating early CNS toxicity. Other, higher concentrations of methanol rapidly raised [Ca 2+] i upwards of 60% over basal resting levels. These result suggest that methanol-induced cerebral vasospasm is a consequence of large rises in intracellular Ca 2+. These events could play a crucial role in methanol-induced cerebral edema, brain hemorrhage, and cerebral and retinal infarcts, eventuating in severe deficits in brain blood flow and the known, subsequent CNS disturbances.

Protein synthesis in presynaptic endings from squid brain: Modulation by calcium ions

Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366–374; Crispino et al. [1993b] J. Neurochem. 61:1144–1146; Crispino et al. [1997] J. Neurosci. 17:7694–7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein. J. Neurosci. Res. 55:776–781, 1999. © 1999 Wiley-Liss, Inc.

Protein synthesis in presynaptic endings from squid brain: modulation by calcium ions.

Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366-374; Crispino et al. [1993b] J. Neurochem. 61:1144-1146; Crispino et al. [1997] J. Neurosci. 17:7694-7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein.

Polydeoxyribonucleotides enhance the proliferation of human skin fibroblasts: Involvement of A 2 purinergic receptor subtypes

It is well-known that nucleotides, nucleosides and purine/pyrimidine bases enhance cell proliferation in vitro. Nevertheless, the molecular mechanisms involved in this mitogenic activity is still controversial, since these compounds are reported both to synergize with growth factor, and to act directly on purinergic receptor inducing per se a proliferative response. It was suggested that cell growth enhancement could be mediated by the A 2 purinergic receptor activation. Here we report that a polydeoxyribonucleotide (PDRN) and adenosine are able to increase, the growth rate of human skin fibroblasts in primary cultures. The proliferative activity exerted by PDRN was significantly counteracted by the A 2 antagonist 3, 7-Dimethyl-1-propijrgylxanthine (DMPX), but not by the A 1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine (PD 116,948, DPCPX). Accordingly, the trophic action of PDRN was mimicked by the A 2 agonist N 6-[2-(3,5-Dimethoxyphenyl)-2-(methylphenyl)-ethyl] adenosine (DPMA), while the A 1 agonist N 6-Cyclopenthyladenosine (CPA) did not show any effect. In microfluorimetric studies, we observed that PDRN and adenosine increased the concentration of cytosolic calcium ions. The PDRN-evoked calcium rise was dose-dependent and DMPX sensitive. Taken together, our results suggest that PDRN may operate as a pro-drug providing the cultured cells with an effective amount of mitogenic deoxyribonucleotides, deoxyribonucleosides and bases; moreover, cell proliferation enhancement that has been induced by PDRN seems to be mediated, at least in part, by the activation of purinergic receptors of the A 2 subtype.

The Role of Protein Kinase C and [Ca2+]i in Superoxide Anion Synthesis and Myeloperoxidase Degranulation of Human Neutrophils.

A chemiluminescence procedure has been developed to determine superoxide anion (O2-) generation and myeloperoxidase (MPO) release from human activated neutrophils. By using this procedure, the role of protein kinase C (PKC) and cytosolic calcium ion (Ca2+[i) for O2- generation and MPO release was examined. Activation of Fc gamma R on neutrophils with IgG-coated zymosan (IgGZ) caused a transient rise of Ca2+[i, followed by O2- generation and MPO release. A PKC inhibitor suppressed completely the O2- generation and slightly the MPO release. Direct activation of PKC by a specific PKC activator, phorbol myristate acetate (PMA), induced a remarkable O2- generation and a small MPO release, indicating that PKC may regulate entirely O2- synthesis and partially MPO degranulation. Influx of extracellular Ca2+ induced by the calcium ionophore A23187 provoked MPO release only. Complete inhibition of this MPO release with a Ca2+/calmodulin (CaM)-coupling inhibitor and a CaM inhibitor provides evidence that Ca2+[i may regulate MPO degranulation through direct activation of CaM, but not PKC. The Ca2+/CaM inhibitors significantly prevented IgGZ-induced O2- generation and MPO release, while they did not affect PMA-induced O2- generation and MPO release. These results suggest that in Fc gamma R-stimulated neutrophils, Ca2+[i activates CaM, which in turn mediates not only activation of PKC-induced O2- synthesis and MPO degranulation, but also MPO degranulation without PKC intermediate.

Blue light but not red light induces a calcium transient in the moss Physcomitrella patens (Hedw.) B., S. &amp; G.

In caulonemal filaments of the moss, Physcomitrella patens, which had been incubated in darkness, 3 s irradiation with blue light (λmax 450 nm) at fluence rates of 100 μmol m−2 s−1 and above caused a transient␣increase in cytosolic calcium ion concentration, [Ca2+]cyt, which was both intensity- and time-dependent. Measurements of [Ca2+]cyt were made using moss transformed with the cDNA for apoaequorin and reconstituting the Ca2+-dependent photoprotein aequorin in the cytosol by incubation in coelenterazine.␣In response to blue light at fluence rates of 100–1000 μmol photons m−2 s−1, [Ca2+]cyt increased transiently from a basal level of approximately 50 nM to between 200 and 700 nM. Irradiation with red light did not evoke any measurable change in [Ca2+]cyt. The presence of calcium in the incubating medium was not required for the increase in [Ca2+]cyt to occur. A mutant strain, gad-139, was identified which required an irradiance of only 1 s to evoke a response. The kinetics showed a delay of approximately 6 s from the beginning of illumination before the beginning of the increase in [Ca2+]cyt. The data suggest that the activation of a photoreceptor rather than the direct opening of calcium channels is involved in this blue-light response.