Wim J.J.M. Scheenen

Besides affecting intracellular calcium signaling, 2-APB reversibly blocks gap junctional coupling in confluent monolayers, thereby allowing measurement of single-cell membrane currents in undissociated cells

2‐Aminoethoxydiphenyl borate (2‐APB) has been widely used as a blocker of the IP3 receptor and TRP channels, including store‐operated calcium channels. We now show in monolayers of normal rat kidney cells (NRK/49F) that 2‐APB completely and reversibly blocks gap junctional intercellular communication at concentrations similar to that required for inhibition of PGF2α‐induced increases in intracellular calcium. Gap junctional conductances between NRK cells were estimated with single‐electrode patch‐clamp measurements and were fully blocked by 2‐APB (50 µM), when applied extracellularly but not via the patch pipette. Half maximal inhibition (IC50) of electrical coupling in NRK cells was achieved at 5.7 µM. Similar results were obtained for human embryonic kidney epithelial cells (HEK293/tsA201) with an IC50 of 10.3 µM. Using 2‐APB as an electrical uncoupler of monolayer cells, we could thus measure inward rectifier potassium, L‐type calcium, and calcium‐dependent chloride membrane currents in confluent NRK monolayers, with properties similar to those in dissociated NRK cells in the absence of 2‐APB. The electrical uncoupling action described here is a new 2‐APB property that promises to provide a powerful pharmacological tool to study single‐cell properties in cultured confluent monolayers and intact tissues by electrical and chemical uncoupling of the cells without the need of prior dissociation.

Monitoring neurotransmitter release using surface-enhanced Raman spectroscopy

Surface-enhanced Raman spectroscopy (SERS) is a promising tool to monitor neurotransmitter release at the single-cell level: it is a sensitive technique that provides structural information of the released compounds and spatial information about their release sites. In this study we demonstrate that depolarization-evoked catecholamine secretion by rat phaeochromocytoma (PC12) cells can be spatially resolved by SERS using silver colloids. A suitable SERS substrate was created by adding silver colloids to the cell culture medium. Nomarski-DIC microscopy combined with reflection confocal laser scanning microscopy showed that the colloids were primarily present on top of the cell membrane. The SERS spectra were successfully corrected for the contribution of cell constituents. Dopamine and noradrenaline were localized by examining the correlation coefficient between spectra and reference catecholamine spectra. Potential improvements of the temporal resolution of the technique are discussed.

Epigenetics: DNA demethylation promotes skeletal myotube maturation

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation‐contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 μM of the DNA methylation inhibitor 5‐azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up‐regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC‐treated myotubes displayed a mature, well‐defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 μM ryanodine, and 100 μM nifedipine, suggesting that action potential‐induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.—Hupkes, M., Jonsson, M. K. B., Scheenen, W. J., van Rotterdam, W., Sotoca, A. M., van Someren, E. P., van der Heyden, M. A. G., van Veen, T. A., van Ravestein‐van Os, R. I., Bauerschmidt, S., Piek, E., Ypey, D. L., van Zoelen, E. J., Dechering, K. J. Epigenetics: DNA demethylation promotes skeletal myotube maturation. FASEB J. 25, 3861–3872 (2011). www.fasebj.org

Spatial and temporal aspects of Ca2+ oscillations in Xenopus laevis melanotrope cells.

Spatio-temporal aspects of Ca2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker omega-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca2+ oscillation is not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca(2+)-wave starting at the cell membrane and going through the cytoplasm at a speed of 33.3 +/- 4.3 microns/s. Before the Ca(2+)-wave enters the nucleus, a delay of 120.0 +/- 24.1 ms occurred. In the nucleus, the speed of a wave was 80.0 +/- 3.0 microns/s. Treatment with the Ca(2+)-ATPase inhibitor thapsigargin (1 MicroM) almost completely eliminated the apparent difference in the basal [Ca2+] in the cytoplasm and the nucleus, reduced the delay of a Ca(2+)-wave before entering the nucleus to 79.8 +/- 8.7 ms, and diminished the nuclear wave speed to 35.0 +/- 4.9 microns/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca2+ before the Ca2+ wave enters the nucleus. After sensitizing IP3 receptors by thimerosal (10 microM) the speed of the cytoplasmic Ca(2+)-wave was increased to 70.3 +/- 3.6 microns/s, suggesting that IP3 receptors may be involved in the propagation of the cytoplasmic Ca2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca2+ oscillations is a complex event involving influx of Ca2+ through N-type Ca2+ channels, propagation of the cytoplasmic Ca2+ wave through mobilization of intracellular stores and a regulated Ca2+ entry into the nucleus. We propose that Ca(2+)-binding proteins may act as a Ca2+ store for propagation of the wave in the nucleus.

Action of stimulatory and inhibitory α-MSH secretagogues on spontaneous calcium oscillations in melanotrope cells of Xenopus laevis

The secretion of α-melanophore-stimulating hormone (α-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca2+ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca2+ ([Ca2+]i) in the melanotrope cell, we have examined the action of well known α-MSH secretagogues on the Ca2+ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine (γ-aminobutyric acid, GABAA agonist), baclofen (GABAB agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca2+ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca2+ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca2+ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.

Opposite effects of T- and L-type Ca2+ channels blockers in generalized absence epilepsy

The role of the T-type Ca(2+) channel blocker, ethosuximide, the L-type Ca(2+) channel blocker, nimodipine and L-type Ca(2+) channel opener, BAY K8644 (1,4 Dihydro-2, 6-dimethyl-5-nitro-4-[trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester), was investigated on spike-wave discharges in WAG/Rij rats. This strain is considered as a genetic model for generalized absence epilepsy. A dose-dependent decrease in the number of spike-wave discharges was found after i.c.v. ethosuximide, an increase after i.p. nimodipine and a decrease after i.c.v. BAY K8644. BAY K8644 was also able to antagonise the effects of nimodipine. Preliminary data were obtained with two conotoxins, MVIIC and GVIA, which block P/Q-type and N-type Ca(2+) channels, respectively. Only after i.c.v. administration of omega-conotoxin GVIA were the number and duration of spike-wave discharges reduced, but animals showed knock-out lying. The latter suggests behavioural or toxic effects and that the decrease in spike-wave activity cannot unequivocally be attributed to blockade of N-type Ca(2+) channels. It can be concluded that T- and L-type Ca(2+) channel blockers show opposite effects on spike-wave discharges. Furthermore, these effects are difficult to explain in terms of a model for spindle burst activity in thalamic relay cells proposed by McCormick and Bal [Sleep and arousal: thalamocortical mechanisms.

Spontaneous calcium oscillations in Xenopus laevis melanotrope cells are mediated by ω-conotoxin sensitive calcium channels

The dynamics of intracellular Ca2+ signalling in single melanotrope cells of the pituitary gland of the amphibian Xenopus laevis have been studied by means of a digital imaging technique using the fluorescent dye Fura-2. When placed in vitro, the majority of the cells (77%) displayed spontaneous oscillatory changes in the free cytosolic Ca2+ concentration with a frequency of 1 +/- 0.25 (SD) min-1. The oscillations rapidly stopped when extracellular Ca2+ was reduced to nanomolar concentrations, revealing their complete dependence on Ca2+ influx. The fact that the Ca2+ oscillations were blocked by 1 microM omega-conotoxin, but not by nifedipine, at concentrations up to 50 microM, indicated that Ca2+ entered the cell via N-type rather than L-type voltage operated Ca2+ channels. Thapsigargin, a putative inhibitor of intracellular Ca(2+)-ATPase activity, elevated the baseline Ca2+ concentration but had no effect on the occurrence of the spontaneous oscillations. This suggests that intracellular Ca2+ pools are not involved in the mechanism underlying spontaneous Ca2+ oscillations. This is the first report showing spontaneous Ca2+ oscillations mediated by N-type Ca2+ channels in melanotrope cells.

Differential responses of corticotropin-releasing factor and urocortin 1 to acute pain stress in the rat brain

It has been hypothesized that corticotropin-releasing factor (CRF) and its related neuropeptide urocortin 1 (Ucn1) play different roles in the initiation and adaptive phases of the stress response, which implies different temporal dynamics of these neuropeptides in response to stressors. We have tested the hypothesis that acute pain stress (APS) differentially changes the dynamics of CRF expression in the paraventricular nucleus of the hypothalamus (PVN), oval subdivision of the bed nucleus of the stria terminalis (BSTov) and central amygdala (CeA), and the dynamics of Ucn1 expression in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW). Thirty minutes after APS, induced by a formalin injection into the left hind paw, PVN, BSTov, CeA and npEW all showed a peak in cFos mRNA expression that was followed by a robust increase in cFos protein-immunoreactivity, indicating a rapid increase in (immediate early) gene expression in all four brain nuclei. CRF-dynamics, however, were affected by APS in a brain nucleus-specific way: in the PVN, CRF-immunoreactivity was minimal at 60 min after APS and concomitant with a marked increase in plasma corticosterone, whereas in the BSTov not CRF peptide but CRF mRNA peaked at 60 min, and in the CeA a surge of CRF peptide occurred as late as 240 min. The npEW differed from the other centers, as Ucn1 mRNA and Ucn1 peptide peaked at 120 min. These results support our hypothesis that each of the four brain centers responds to APS with CRF/Ucn1 dynamics that are specific as to nature and timing. In particular, we propose that CRF in the PVN plays a major role in the initiation phase, whereas Ucn1 in the npEW may act in the later, termination phase of the adaptation response to APS.

Ca2+ oscillations in melanotropes of Xenopus laevis: their generation, propagation, and function

The melanotrope cell of the amphibian Xenopus laevis is a neuroendocrine transducer that converts neuronal input concerning the color of background into an endocrine output, the release of alpha-melanophore-stimulating hormone (alpha-MSH). The cell displays intracellular Ca(2+) oscillations that are thought to be the driving force for secretion as well as for the expression of genes important to the process of background adaptation. Here we review the functioning of the Xenopus melanotrope cell, with emphasis on the role of Ca(2+) oscillations in signal transduction in this cell. We start by giving a general overview of the evolution of Ca(2+) as an intracellular messenger molecule. This is followed by an examination of the melanotrope as a neuroendocrine integrator cell. Then, the evidence that Ca(2+) oscillations drive the secretion of alpha-MSH is reviewed, followed by a similar analysis of the evidence that the same oscillations regulate the expression of proopiomelanocortin (POMC), the precursor protein for alpha-MSH. Finally, the possible importance of the pattern of Ca(2+) signaling to melanotrope cell function is considered.

Multiple control and dynamic response of the Xenopus melanotrope cell.

Some amphibian brain-melanotrope cell systems are used to study how neuronal and (neuro)endocrine mechanisms convert environmental signals into physiological responses. Pituitary melanotropes release alpha-melanophore-stimulating hormone (alpha-MSH), which controls skin color in response to background light stimuli. Xenopus laevis suprachiasmatic neurons receive optic input and inhibit melanotrope activity by releasing neuropeptide Y (NPY), dopamine (DA) and gamma-aminobutyric acid (GABA) when animals are placed on a light background. Under this condition, they strengthen their synaptic contacts with the melanotropes and enhance their secretory machinery by upregulating exocytosis-related proteins (e.g. SNAP-25). The inhibitory transmitters converge on the adenylyl cyclase system, regulating Ca(2+) channel activity. Other messengers like thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH, from the magnocellular nucleus), noradrenalin (from the locus coeruleus), serotonin (from the raphe nucleus) and acetylcholine (from the melanotropes themselves) stimulate melanotrope activity. Ca(2+) enters the cell and the resulting Ca(2+) oscillations trigger alpha-MSH secretion. These intracellular Ca(2+) dynamics can be described by a mathematical model. The oscillations travel as a wave through the cytoplasm and enter the nucleus where they may induce the expression of genes involved in biosynthesis and processing (7B2, PC2) of pro-opiomelanocortin (POMC) and release (SNAP-25, munc18) of its end-products. We propose that various environmental factors (e.g. light and temperature) act via distinct brain centers in order to release various neuronal messengers that act on the melanotrope to control distinct subcellular events (e.g. hormone biosynthesis, processing and release) by specifically shaping the pattern of melanotrope Ca(2+) oscillations.