Ultrafast Plasmon Dynamics in Crystalline LiF Triggered by Intense Extreme UV Pulses.

An extreme ultraviolet pump and visible-light probe transmission experiment in crystalline LiF, carried out at the Free Electron Laser facility FERMI, revealed an oscillating time dependence of the plasmon mode excited in the high-density high-temperature electron plasma. The effect is interpreted as a fingerprint of the electron-ion interaction: the ion motion, shaped by the electron dynamic screening, induces, in turn, electron density fluctuations that cause the oscillation of the plasmon frequency at the timescale of the ion dynamics. Fitting the high resolution transmission data with an RPA model for the temperature-dependent dielectric function, which includes electron self-energy and electron-ion coupling, confirms the interpretation of the time modulation of the plasmon mode.

Raman spectroscopy of graphene under ultrafast laser excitation.

The equilibrium optical phonons of graphene are well characterized in terms of anharmonicity and electron-phonon interactions; however, their non-equilibrium properties in the presence of hot charge carriers are still not fully explored. Here we study the Raman spectrum of graphene under ultrafast laser excitation with 3 ps pulses, which trade off between impulsive stimulation and spectral resolution. We localize energy into hot carriers, generating non-equilibrium temperatures in the ~1700-3100 K range, far exceeding that of the phonon bath, while simultaneously detecting the Raman response. The linewidths of both G and 2D peaks show an increase as function of the electronic temperature. We explain this as a result of the Dirac cones' broadening and electron-phonon scattering in the highly excited transient regime, important for the emerging field of graphene-based photonics and optoelectronics.

Folate-based single cell screening using surface enhanced Raman microimaging.

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostics.

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes.

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.

Ca2+ depletion from granules inhibits exocytosis. A study with insulin-secreting cells.

The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that the size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophore followed by agents that collapse acidic pH gradients. In the present study we have employed this protocol in the insulin-secreting cell line Ins-1 to determine whether the Ca2+ trapped in the secretory granules plays a role in exocytosis. The results demonstrate that a high proportion of ionophore-mobilizable Ca2+ in Ins-1 cells resides in the acidic compartment. The latter pool, however, does not significantly contribute to the [Ca2+]i changes elicited by thapsigargin and the inositol trisphosphate-producing agonist carbachol. By monitoring membrane capacitance at the single cell level or by measuring insulin release in cell populations, we show that Ca2+ mobilization from nonacidic Ca2+ pools causes a profound and long lasting increase in depolarization-induced secretion, whereas breakdown of granule pH had no significant effect. In contrast, releasing Ca2+ from the acidic pool markedly reduces secretion. It is suggested that a high Ca2+ concentration in the secretory compartment is needed to sustain optimal exocytosis.

Store depletion triggers the calcium release-activated calcium current (ICRAC) in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell lines.

In endothelial cells, different types of Ca2+ conductances have been described, but none of them has been clearly identified as ICRAC, the Ca2+ release-activated Ca2+ current originally described in mast and lymphoma cells. Here we show that in bovine pulmonary artery endothelial cells (CPAE) depletion of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate (InsP3), Ca2+ ionophores and Ca2+ pump inhibitors activates a Ca2+-selective conductance in the presence of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA). The current shows inward rectification, a highly positive reversal potential and is blocked by micromolar concentrations of La3+. The conditions used in studies of endothelial cells were also employed in those of HEK-293, an embryonic kidney cell line commonly used to express putative store-operated channels, and Jurkat cells, the reference cell model. Similar to CPAE, HEK cells also have an ICRAC-like current. At 0 mV holding potential the estimated current density is -0.1 and -0.2 pA/pF in CPAE and HEK cells respectively, i.e. 15 and 30% of that measured in Jurkat cells. As shown in studies of Jurkat cells, larger Na+ currents are detectable in CPAE and HEK cells following store depletion in Ca2+- and Mg2+-free medium. The current carried by Na+ ions is similarly blocked by micromolar La3+, is inwardly rectifying and has a positive reversal potential.

Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1 cells.

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+].

ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca2+ chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca2+] within the ER can be lowered and restored to its original level within 10-15 s without modifications of Ca2+ pumps or release channels. Using these new tools, we demonstrate here that Ca2+ release-activated Ca2+ current (ICRAC) is activated (a) solely by reduction of free [Ca2+] within the ER and (b) by any measurable decrease in [Ca2+]ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.

Glucose-induced insulin secretion in INS-1 cells depends on factors present in fetal calf serum and rat islet-conditioned medium.

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (-78, -68, and -56%, respectively), whereas the response to 30 mmol/l K+ was only slightly decreased. In these cells: 1) nutrient metabolism assessed by tetrazolium salt reduction was reduced in response to pyruvate and leucine, which are mainly metabolized in the mitochondria; 2) oxidation of both [3,4-(14)C]glucose and [1-(14)C]pyruvate was decreased (-22 and -32%, respectively); 3) glucose failed to depolarize the membrane potential, whereas tolbutamide was fully active; 4) video imaging analysis of cytosolic Ca2+ showed a decrease in the population of glucose-responsive cells, while the response to 30 mmol/l K+ was preserved; 5) serum replenishment for 3 days restored glucose-induced insulin secretion. Interestingly, conditioned serum-free medium from rat islets maintained the insulin secretory function of INS-1 cells, although glucagon, somatostatin, and some other factors failed to restore the function. In contrast, conditioned media from HepG2, PC12, and human umbilical vein endothelial cells did not substitute for serum. Thus, the impaired insulin secretion of the cells cultured in the defined medium is best explained by defective mitochondrial metabolism. Islet cells, but not INS-1 cells, produce factors required for normal signal generation by nutrient secretagogues.

Dynamic properties of an inositol 1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines.

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.

Intracellular ADP modulates the Ca2+ release-activated Ca2+ current in a temperature- and Ca2+-dependent Way.

The rat basophilic cell line RBL-1 is known to express high levels of the Ca2+ current activated by store depletion, known as Ca2+ release-activated Ca2+ current (ICRAC), the main Ca2+ influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca2+ influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between ICRAC and cell metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces ICRAC induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of ICRAC, since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca2+ buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis. Moreover, the inhibition is specific for ADP and is partially mimicked by ADPbetaS and AMP, but not by GDP or GTP.

Stimulation of single L-type calcium channels in rat pituitary GH3 cells by thyrotropin-releasing hormone.

Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.

Receptor-activated Ca2+ influx: how many mechanisms for how many channels?

Receptors that are coupled to the production of inositol (1,4,5)-trisphosphate cause an increase in cytosolic free Ca2+ concentration as a consequence of both Ca2+ mobilization from intracellular stores and Ca2+ influx through the plasma membrane. Although this latter phenomenon appears attributable to the activation of a number of Ca(2+)-permeable channels, the channels that are controlled by the Ca2+ content of the intracellular stores have recently received much attention. In this review, Cristina Fasolato, Barbara Innocenti and Tullio Pozzan summarize the characteristics of this Ca(2+)-influx pathway and discuss the hypotheses about its mechanism of activation and its relationship with other receptor-activated Ca2+ channels.

A GTP-dependent step in the activation mechanism of capacitative calcium influx.

Calcium influx in electrically non-excitable cells is regulated by the filling state of intracellular calcium stores. Depletion of stores activates plasma membrane channels that are voltage-independent and highly selective for Ca2+ ions. We report here that the activation of plasma membrane Ca2+ currents induced by depletion of Ca2+ stores requires a diffusible cytosolic factor that washes out with time when dialyzing cells in the whole-cell configuration of the patch-clamp technique. The activation of calcium release-activated calcium current (ICRAC) by ionomycin- or inositol 1,4,5-trisphosphate-induced store depletion is blocked by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and guanyl-5'-yl imidodiphosphate, non-hydrolyzable analogs of GTP, suggesting the involvement of a GTP-binding protein. The inhibition by GTP gamma S occurs at a step prior to the activation of ICRAC and is prevented by the addition of GTP. We conclude that the activation mechanism of depletion-induced Ca2+ influx encompasses a GTP-dependent step, possibly involving an as yet unidentified small GTP-binding protein.

Calcium influx and its control by calcium release.

Changes in the concentration of intracellular Ca2+ are crucial for signal transduction in virtually every cell. In the past year, more of the diversity of receptor-mediated Ca2+ influx mechanisms has been shown, and it has been disclosed that one of the most effective Ca2+ influx pathways, known as 'capacitative Ca2+ entry', occurs via Ca(2+)-selective ion channels in the plasma membrane that are activated following depletion of intracellular Ca2+ stores. Although the exact activation mechanism of capacitative Ca2+ entry still remains a mystery, the identification of plasma membrane currents following store depletion and the characterization of their biophysical properties opens the possibility of unraveling the features and molecular components of the phenomenon of capacitative Ca2+ entry.

Multiple mechanisms of manganese-induced quenching of fura-2 fluorescence in rat mast cells.

Whole-cell patch-clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study Mn2+ influx in rat peritoneal mast cells. The calcium-selective current, activated by depletion of intracellular calcium stores (ICRAC for calcium release-activated calcium current), supports a small but measurable Mn2+ current. In the presence of intracellular BAPTA, a Mn2+ current through ICRAC was recorded in isotonic MnCl2 (100 mM) without a significant quenching of fura-2 fluorescence. Its amplitude was 10% of that measured in physiological solution containing 10 mM Ca2+. However, following store depletion, a significant quenching of fura-2 fluorescence could be measured only when intracellular BAPTA was omitted, so that all the incoming Mn2+ could be captured by the fluorescent dye. Two other ionic currents activated by receptor stimulation also induced Mn2+ quenching of fura-2 fluorescence: a small current through non-specific cation channels of 50-pS unitary conductance and a distinct cationic current of large amplitude. In addition to these influx mechanisms, Mn2+ was taken up into calcium stores and was subsequently co-released with Ca2+ by Ca(2+)-mobilizing agonists.

Ca2+ and Mn2+ influx through receptor-mediated activation of nonspecific cation channels in mast cells.

Whole-cell patch-clamp recordings of membrane currents and Fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study calcium influx through receptor-activated cation channels in rat peritoneal mast cells. Cation channels were activated by the secretagogue compound 48/80, whereas a possible concomitant Ca2+ entry through pathways activated by depletion of calcium stores was blocked by dialyzing cells with heparin. Heparin effectively suppressed the transient Ca2+ release induced by 48/80 and abrogated inositol 1,4,5-trisphosphate-induced calcium influx without affecting activation of 50-pS cation channels. There was a clear correlation between changes in [Ca2+]i and the activity of 50-pS channels. The changes in [Ca2+]i increased with elevation of extracellular Ca2+. At the same time, inward currents through 50-pS channels were diminished as more Ca2+ permeated. This effect was due to a decrease in slope conductance and a reduction in the open probability of the cation channels. In physiological solutions, 3.6% of the total current was carried by Ca2+. The cation channels were not only permeable to Ca2+ but also to Mn2+, as evidenced by the quench of Fura-2 fluorescence. Mn2+ current through 50-pS channels could not be resolved at the single-channel level. Our results suggest that 50-pS cation channels partially contribute to sustained increases of [Ca2+]i in mast cells following receptor activation.

Receptor-activated Ca2+ influx. Two independently regulated mechanisms of influx stimulation coexist in neurosecretory PC12 cells.

Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.