Oscillations In Intracellular Calcium Concentration Research Articles

Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

Aims/HypothesisStudies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.MethodsCells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.ResultsGlucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Conclusions/InterpretationFunctions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

High phosphate reduces host ability to develop arbuscular mycorrhizal symbiosis without affecting root calcium spiking responses to the fungus.

The arbuscular mycorrhizal symbiosis associates soil fungi with the roots of the majority of plants species and represents a major source of soil phosphorus acquisition. Mycorrhizal interactions begin with an exchange of molecular signals between the two partners. A root signaling pathway is recruited, for which the perception of fungal signals triggers oscillations of intracellular calcium concentration. High phosphate availability is known to inhibit the establishment and/or persistence of this symbiosis, thereby favoring the direct, non-symbiotic uptake of phosphorus by the root system. In this study, Medicago truncatula plants were used to investigate the effects of phosphate supply on the early stages of the interaction. When plants were supplied with high phosphate fungal attachment to the roots was drastically reduced. An experimental system was designed to individually study the effects of phosphate supply on the fungus, on the roots, and on root exudates. These experiments revealed that the most important effects of high phosphate supply were on the roots themselves, which became unable to host mycorrhizal fungi even when these had been appropriately stimulated. The ability of the roots to perceive their fungal partner was then investigated by monitoring nuclear calcium spiking in response to fungal signals. This response did not appear to be affected by high phosphate supply. In conclusion, high levels of phosphate predominantly impact the plant host, but apparently not in its ability to perceive the fungal partner.

Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms

Inhalation of (nano)particles may lead to pulmonary inflammation. However, the precise mechanisms of particle uptake and generation of inflammatory mediators by alveolar macrophages (AM) are still poorly understood. The aim of this study was to investigate the interactions between particles and AM and their associated pro-inflammatory effects in relation to particle size and physico-chemical properties.NR8383 rat lung AM were treated with ultrafine (uf), fine (f) TiO2 or fine crystalline silica (DQ12 quartz). Physico-chemical particle properties were investigated by transmission electron microscopy, elemental analysis and thermogravimetry. Aggregation and agglomeration tendency of the particles were determined in assay-specific suspensions by means of dynamic light scattering.All three particle types were rapidly taken up by AM. DQ12 and ufTiO2 , but not fTiO2 , caused increased extracellular reactive oxygen species (ROS), heme oxygenase 1 (HO-1) mRNA expression and tumor necrosis factor (TNF)-α release. Inducible nitric oxide synthase (iNOS) mRNA expression was increased most strongly by ufTiO2 , while DQ12 exclusively triggered interleukin (IL) 1β release. However, oscillations of intracellular calcium concentration and increased intracellular ROS were observed with all three samples. Uptake inhibition experiments with cytochalasin D, chlorpromazine and a Fcγ receptor II (FcγRII) antibody revealed that the endocytosis of fTiO2 by the macrophages involves actin-dependent phagocytosis and macropinocytosis as well as clathrin-coated pit formation, whereas the uptake of ufTiO2 was dominated by FcγIIR. The uptake of DQ12 was found to be significantly reduced by all three inhibitors. Our findings suggest that the contrasting AM responses to fTiO2 , ufTiO2 and DQ12 relate to differences in the involvement of specific uptake mechanisms.

Kisspeptin-10 Facilitates a Plasma Membrane-Driven Calcium Oscillator in Gonadotropin-Releasing Hormone-1 Neurons

Kisspeptins, the natural ligands of the G-protein-coupled receptor (GPR)-54, are the most potent stimulators of GnRH-1 secretion and as such are critical to reproductive function. However, the mechanism by which kisspeptins enhance calcium-regulated neuropeptide secretion is not clear. In the present study, we used GnRH-1 neurons maintained in mice nasal explants to examine the expression and signaling of GPR54. Under basal conditions, GnRH-1 cells exhibited spontaneous baseline oscillations in intracellular calcium concentration ([Ca(2+)](i)), which were critically dependent on the operation of voltage-gated, tetrodotoxin (TTX)-sensitive sodium channels and were not coupled to calcium release from intracellular pools. Activation of native GPR54 by kisspeptin-10 initiated [Ca(2+)](i) oscillations in quiescent GnRH-1 cells, increased the frequency of calcium spiking in oscillating cells that led to summation of individual spikes into plateau-bursting type of calcium signals in a subset of active cells. These changes predominantly reflected the stimulatory effect of GPR54 activation on the plasma membrane oscillator activity via coupling of this receptor to phospholipase C signaling pathways. Both components of this pathway, inositol 1,3,4-trisphosphate and protein kinase C, contributed to the receptor-mediated modulation of baseline [Ca(2+)](i) oscillations. TTX and 2-aminoethyl diphenylborinate together abolished agonist-induced elevation in [Ca(2+)](i) in almost all cells, whereas flufenamic acid was less effective. Together these results indicate that a plasma membrane calcium oscillator is spontaneously operative in the majority of prenatal GnRH-1 neurons and is facilitated by kisspeptin-10 through phosphatidyl inositol diphosphate hydrolysis and depolarization of neurons by activating TTX-sensitive sodium channels and nonselective cationic channels.

Effect of cyclic guanosine‐monophosphate on porcine retinal vasomotion

Vasomotion refers to periodic oscillations in vascular tone that ensure the intermittent supply of blood to adjacent microvascular units. Previous evidence from vessels outside the eye suggests that cyclic guanosine-monophosphate (cGMP) is involved in the regulation of vasomotion, but it is unknown whether this compound has an effect on vasomotion in retinal vessels. Retinal arterioles from porcine eyes were studied in a wire myograph. After initiation of vasomotion, the vessels were stimulated with increasing concentrations of the cGMP agonist 8-Br-cGMP (n = 6), the phosphodiesterase inhibitor zaprinast (n = 6) and the cGMP synthesis inhibitor L-NAME (n = 6). High concentrations of L-NAME blocked vasomotion, and control experiments (n = 20) using 8-Br-cGMP, S-nitroso-N-acetylpenicillamine (SNAP), adenosine and pinacidil were carried out to elucidate whether this effect was related to changes in the general tone of the vessel. Additionally, the relationship between oscillations in vascular tone and intracellular calcium concentration was studied. Induction of cGMP agonistic activity with either 8-Br-cGMP or zaprinast lowered the vasomotion frequency significantly, whereas L-NAME-induced inhibition of cGMP increased this frequency. Neither of the agents affected the amplitude of the oscillations. The control experiments indicated that the effect of cGMP on vasomotion frequency was independent of the accompanying increase in tone. The oscillations in tone during vasomotion were accompanied by similar oscillations in intracellular calcium concentration. Cyclic GMP lowers the frequency without affecting the amplitude of vasomotion in isolated porcine retinal arterioles.

Oscillatory juxtaglomerular tubular cells – a possible new tubule‐to‐arteriole signaling pathway

Tubular segments distal (downstream) and proximal (upstream) to the macula densa are closely associated with the afferent and efferent arterioles, respectively. The epithelial cells in the perimeter of the macula densa demonstrate spontaneous oscillations in intracellular calcium concentration ([Ca2+]i). We hypothesized that intracellular Ca2+ signaling in the tubular segment proximal to the macula densa might be associated with intracellular Ca2+ changes in the adjacent efferent arteriole. We utilized isolated perfused cortical thick ascending limb (cTAL) with attached glomerulus-efferent arteriole and assessed [Ca2+]i in the tubular and vascular elements with widefield fluorescence microscopy using fura-2 or multiphoton excitation fluorescence microscopy and 4-dimensional volume-rendering. Here we report that the vascular smooth muscle cells of the efferent arteriole and the epithelial cells of the adjacent cTAL demonstrate synchronous oscillations or spikes in [Ca2+]i. Interestingly, the initiation of an oscillation in [Ca2+]i in the efferent arteriole required [Ca2+]i in the tubular epithelium to reach an apparent threshold. Dissection of the efferent arteriole away from the cTAL reduced the amplitude of oscillations in the efferent arteriole. Also, luminal administration of furosemide, an inhibitor of the Na+:2Cl−:K+ cotransporter, blocked the amplitude of oscillations in the epithelial cells of the cTAL by 53 %, hydrochlorothiazide from the lumen had no effect. These findings suggest that the tubular segment downstream from the macula densa possess oscillations in [Ca2+]i which may play a role in signaling to the efferent arteriole.

Close Encounters Between the Na Pump and the IP 3 R

Miyakawa-Naito et al. uncovered an intriguing interaction between the Na,K-ATPase (Na + - and K + -dependent adenosine triphosphatase) and the inositol 1,4,5-trisphosphate receptor (IP 3 R), implicating a possible interaction of these two proteins in cell signaling. Ouabain, a cardiac glycoside initially identified as an arrow poison and long used experimentally as an inhibitor of the Na,K-ATPase, has more recently been proposed to function as a hormone. Partial inhibition of the Na,K-ATPase by ouabain elicits oscillations in intracellular calcium concentration ([Ca 2+ ] i ) and stimulates several transcription factors, suggesting that association of ouabain (a steroid) with the plasma membrane Na,K-ATPase activates a cell signaling pathway. Miyakawa-Naito et al. used pharmacological analysis to determine that ouabain-induced [Ca 2+ ] i oscillations in rat renal proximal tubule cells depended on IP 3 R-mediated release of calcium from endoplasmic reticulum (ER) stores. Fluorescence resonance energy transfer (FRET) analysis in COS-7 cells indicated close proximity of Na,K-ATPase to IP 3 R2 and IP 3 R3 and coimmunoprecipitation indicated that the Na,K-ATPase associated with IP 3 R3 in a complex. FRET and coimmunoprecipitation were increased by ouabain and abolished by disruption of the actin cytoskeleton, which also abolished ouabain-induced Ca 2+ oscillations, implicating the cytoskeleton in the association of plasma membrane Na,K-ATPase with ER IP 3 R. Oscillations persisted in cells treated with a phospholipase C inhibitor or transfected with a high-affinity IP 3 -binding protein. Partial truncation of the Na,K-ATPase α-1 subunit N-terminal indicated that the ability of ouabain-stimulated Na,K-ATPase to elicit Ca 2+ oscillations and activate nuclear factor-κB depended on a region that was not required for enzymatic activity. This study thus defines a functional microdomain linking the plasma membrane to ER Ca 2+ stores and adds weight to the possible role of the Na,K-ATPase as a hormone receptor and transducer of cell signaling. A. Miyakawa-Naito, P. Uhlén, M. Lal, O. Aizman, K. Mikoshiba, H. Brismar, S. Zelenin, A. Aperia, Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations. J. Biol. Chem . 278 , 50355-50361 (2003). [Abstract] [Full Text]

Oscillating Kinase Activity

Protein phosphorylation regulates numerous cellular processes. Although the importance of the spatiotemporal context of second messenger activity has become increasingly apparent, the precise consequences of the spatiotemporal dynamics of second messenger signals on the extent, duration, and specificity of activity of downstream kinases has been less clear. Violin et al. developed C kinase activity reporter (CKAR), a genetically encoded reversible fluorescence resonance energy transfer (FRET)-based reporter of protein kinase C (PKC)-mediated phosphorylation. They used CKAR in combination with novel FRET-based reporters of PKC translocation, phospholipase C (PLC) activity, and diacylglycerol (DAG) to study the regulation of PKC-mediated phosphorylation in MDCK and HeLa cells in the context of second messenger, substrate, kinase, and phosphatase activity and localization. Maximal activation of PKC by phorbol esters did not saturate phosphorylation of cytoplasmic CKAR unless phosphatases were also inhibited; when CKAR was targeted to the plasma membrane, however, phosphatase inhibition had little effect. Moreover, histamine, which caused only a slight increase in FRET of cytosolic CKAR, elicited oscillations of membrane-targeted-CKAR phosphorylation in HeLa cells. These oscillations were synchronized with oscillations in intracellular calcium concentration ([Ca2+]i), were not accompanied by oscillations in DAG or in PLC activity, and correlated with PKC translocation to the plasma membrane. In MDCK cells, however, ATP stimulated synchronous increases in [Ca2+]i, membrane-targeted CKAR phosphorylation, PLC activity, and DAG. These data suggest high fidelity of kinase activity to second messenger dynamics.J. D. Violin, J. Zhang, R. Y. Tsien, A. C. Newton, A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899-909 (2003). [Abstract] [Full Text]

Oscillating Kinase Activity

Protein phosphorylation regulates numerous cellular processes. Although the importance of the spatiotemporal context of second messenger activity has become increasingly apparent, the precise consequences of the spatiotemporal dynamics of second messenger signals on the extent, duration, and specificity of activity of downstream kinases has been less clear. Violin et al . developed C kinase activity reporter (CKAR), a genetically encoded reversible fluorescence resonance energy transfer (FRET)-based reporter of protein kinase C (PKC)-mediated phosphorylation. They used CKAR in combination with novel FRET-based reporters of PKC translocation, phospholipase C (PLC) activity, and diacylglycerol (DAG) to study the regulation of PKC-mediated phosphorylation in MDCK and HeLa cells in the context of second messenger, substrate, kinase, and phosphatase activity and localization. Maximal activation of PKC by phorbol esters did not saturate phosphorylation of cytoplasmic CKAR unless phosphatases were also inhibited; when CKAR was targeted to the plasma membrane, however, phosphatase inhibition had little effect. Moreover, histamine, which caused only a slight increase in FRET of cytosolic CKAR, elicited oscillations of membrane-targeted-CKAR phosphorylation in HeLa cells. These oscillations were synchronized with oscillations in intracellular calcium concentration ([Ca2 + ] i ), were not accompanied by oscillations in DAG or in PLC activity, and correlated with PKC translocation to the plasma membrane. In MDCK cells, however, ATP stimulated synchronous increases in [Ca 2+ ] i , membrane-targeted CKAR phosphorylation, PLC activity, and DAG. These data suggest high fidelity of kinase activity to second messenger dynamics. J. D. Violin, J. Zhang, R. Y. Tsien, A. C. Newton, A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161 , 899-909 (2003). [Abstract] [Full Text]

Porcine sperm factor supports activation and development of bovine nuclear transfer embryos.

A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca(2+)](i)) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca(2+)](i) and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca(2+)](i) oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60-90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3-5 h postinjection and a second injection was necessary to extend the [Ca(2+)](i) oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.

CAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel.

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.

Responsiveness of vascular endothelium to shear stress: potential role of ion channels and cellular cytoskeleton (review).

The frictional forces associated with blood flow expose vascular endothelium in arteries to a complex and highly dynamic shear stress distribution. The ability of endothelial cells to respond to shear stress is essential for arterial vasoregulation in response to acute hemodynamic changes and for vascular wall remodeling following chronic changes in blood flow. Furthermore, endothelial responsiveness to shear stress may play a role in the localization of early atherosclerotic lesions. Shear stress elicits a wide range of humoral, metabolic, and structural responses in endothelial cells. These include activation of ion channels and of G proteins, induction of oscillations in intracellular calcium concentration, alterations in the expression of various important genes, and extensive cytoskeletal reorganization. Mechanisms of shear stress sensing and transmission in endothelium are discussed in light of the complex shear stress distribution to which endothelial cells are exposed in vivo and with particular emphasis on the potentially central role of flow-sensitive ion channels and the cellular cytoskeleton. Finally, the ability of endothelial cells to distinguish among and to respond differentially to different types of shear stress is highlighted.

Astrocytes are required for the oscillatory activity in cultured hippocampal neurons.

Synchronous oscillations of intracellular calcium concentration ([Ca2+]i) and of membrane potential occurred in a limited population of glutamatergic hippocampal neurons grown in primary cultures. The oscillatory activity occurred in synaptically connected cells only when they were in the presence of astrocytes. Microcultures containing only one or a few neurons also displayed oscillatory activity, provided that glial cells participated in the network. The glutamate-transporter inhibitors L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC) and dihydrokainate, which produce an accumulation of glutamate in the synaptic microenvironment, impaired the oscillatory activity. Moreover, in neurons not spontaneously oscillating, though in the presence of astrocytes, oscillations were induced by exogenous L-glutamate, but not by the stereoisomer D-glutamate, which is not taken up by glutamate transporters. These data demonstrate that astrocytes are essential for neuronal oscillatory activity and provide evidence that removal of glutamate from the synaptic environment is one of the major mechanisms by which glial cells allow the repetitive excitation of the postsynaptic cell.

L-type Ca2+ Channels and K+ Channels Specifically Modulate the Frequency and Amplitude of Spontaneous Ca2+ Oscillations and Have Distinct Roles in Prolactin Release in GH3 Cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.

On the Structure of the Bursting Region for a Neuronal Model

We examine the question of whether the bursting region of a neuronal model is simply or multiply connected in the parameter space. Taking into account that the presence of slow oscillations of intracellular calcium concentration is a necessary condition for a bursting mode, we analyze the subysystem responsible for the slow component of bursting. Explicit parametric form of the Hopf bifurcation is given. It enables us to reduce the problem to determining the number of the bifurcation curves by finding the number of discontinuities of the expressions giving the bifurcation parameter values. The results obtained suggest that the bursting region is simply connected over the physiologically possible parameter range.

The Two Intracellular Ca2+Release Channels, Ryanodine Receptor and Inositol 1,4,5-Trisphosphate Receptor, Play Different Roles during Fertilization in Ascidians

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs ofPhallusia mammillatausing whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3(10 μMperfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15–20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.

Rhythm generation in organotypic medullary cultures of newborn rats

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO2 and H+. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 μM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 μM) and/or hydroxysaclofen (200 μM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 μM) abolished rhythmicity. (3) While atropine sulphate (5 μM) was ineffective in modulating periodic discharges nicotine (100 μM) — like CO2 — shortened the intervals between the periodic events; hexamethonium (50–100 μM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 μM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10–50 μM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50–100 μM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO2/acidosis. (6) Dantrolene (10 μM), an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 μM) which blocks endoplasmic Ca2+-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)–(7) indicate that transmembrane calcium fluxes as well as intracellular Ca2+-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.

Differential regulation of cytosolic calcium between afferent arteriol ar smooth muscle cells from mouse kidney.

Using a combination of whole-cell patch-clamping and calcium microspectrofluorimetry we have examined the calcium regulation and calcium-activated electrical currents in smooth muscle cells along mouse renal afferent arterioles. In cells located at a distance up to 50microm from the glomerular vascular pole, guanosine 5'-[gamma-thio]triphosphate, (GTP[tau -S], 100micromol/l) and angiotensin II (1micromol/l) evoked internal calcium mobilization and oscillations of intracellular calcium concentration. Membrane depolarization up to +30mV did not increase cytosolic free calcium in these cells, which displayed calcium-activated chloride currents. In cells located more distant than 100microm from the glomerular vascular pole, GTP[tau -S] did not increase cytosolic calcium at negative membrane potentials. Depolarization of these cells to 0mV or positive membrane potentials increased intracellular calcium in a dihydropyridine-sensitive manner. These cells responded to angiotensin II with single calcium transients and also displayed calcium-activated chloride currents. These findings suggest that intracellular calcium is differentially regulated between afferent arteriolar smooth muscle cells from mouse kidney: in smooth muscle cells which are distant from the glomerular vascular pole, cytosolic calcium is increased primarily via calcium influx through potential-operated calcium channels, whilst in cells which are close to the vascular pole intracellular calcium is elevated predominantly via mobilization from internal stores. Both cell types are equipped with receptors for angiotensin II and possess calcium-activated chloride channels.

Early functional neural networks in the developing retina.

In the adult mammalian retina, the principal direction of information flow is along a vertical pathway from photoreceptors to retinal interneurons to ganglion cells, the output neurons of the retina. We report here, however, that initially in development, at a time when the photoreceptors are not yet even present, there are already functionally defined networks within the retina. These networks are spontaneously active rather than visually driven, and they involve horizontal rather than vertical pathways. By means of optical recording using the calcium-sensitive dye Fura-2, we have found that sets of retinal ganglion cells and amacrine cells, a type of retinal interneuron, undergo synchronized oscillations in intracellular calcium concentration. These oscillations are highly correlated among subgroups of neighbouring cells, and spread in a wave-like fashion tangentially across the retina. Thus, in development of retinal circuitry, the initial patterning of neuronal function occurs in the horizontal domain before the adult pattern of vertical information transfer emerges.