Ernst Niggli

Imaging the hierarchical Ca2+ signalling system in HeLa cells.

1. Confocal microscopy was used to investigate hormone‐induced subcellular Ca2+ release signals from the endoplasmic reticulum (ER) in a prototype non‐excitable cell line (HeLa cells). 2. Histamine application evoked two types of elementary Ca2+ signals: (i) Ca2+ blips arising from single ER Ca2+ release channels (amplitude, 30 nM; lateral spreading, 1.3 microns); (ii) Ca2+ puffs resulting from the concerted activation of several Ca2+ blips (amplitude, 170 nM; spreading, 4 microns). 3. Ca2+ waves in the HeLa cells arose from a variable number of initiation sites, but for individual cells, the number and subcellular location of the initiation sites were constant. The kinetics and amplitude of global Ca2+ signals were directly proportional to the number of initiation sites recruited. 4. Reduction of the feedback inherent in intracellular Ca2+ release caused saltatoric Ca2+ waves, revealing the two principal steps underlying wave propagation: diffusion and regeneration. Threshold stimulation evoked abortive Ca2+ waves, caused by the limited recruitment of Ca2+ puffs. 5. The hierarchy of Ca2+ signalling events, from fundamental levels (blips) to intermediate levels (puffs) to Ca2+ waves, is a prototype for Ca2+ signal transduction for non‐excitable cells, and is also analogous to the Ca2+ quarks, Ca2+ sparks and Ca2+ waves in cardiac muscle cells.

Ratiometric confocal Ca2+-measurements with visible wavelength indicators in isolated cardiac myocytes

Abstract We present a new method for ratiometric Ca2+ measurements using indicators with excitation spectra in the visible range of wavelengths. Laser-scanning confocal microscopy was used to record intracellular Ca2+-signals with high temporal and spatial resolution in single cardiac myocytes. The patch-clamp technique was applied to load the cells with the fluorescent Ca2+-indicators and to follow the membrane currents with the fluorescence signals simultaneously. Intracellular free Ca2+-concentration ([Ca2+]i) was estimated with a ratiometric method. An in vitro calibration procedure was used to convert the fluorescence ratio obtained with two different Ca2+-indicators (Fluo-3 and Fura-Red) into Ca2+-concentrations. Fluo-3 showed an increase in fluorescence upon a rise in intracellular Ca2+-concentration, while the Fura-Red fluorescence decreased. Since the fluorescence of Fluo-3 was around 2-fold brighter than the Fura-Red signal the cells were loaded with a 1:2 mixture of the two indicators. The large increase of the fluorescence ratio during a rise in [Ca2+]i (up to 4-fold) allowed us to record time-resolved signals with this mixture even when monitored in a very small subcellular volume (around 1 μm3). Long lasting continuous recordings of the fluorescence were possible because the dye-mixture exhibited no detectable bleaching with illumination periods of up to 30 s. The use of the Fluo-3 Fura-Red ratio method should significantly facilitate and improve quantitative measurements of [Ca2+]i with high temporal and spatial resolution. Moreover, this approach is especially valuable when used with confocal microscopes which are usually equipped with lasers in the visible light range. Furthermore, it may be possible to use the same approach with mixtures of other indicators to estimate the concentration of other biologically important ions/compounds with a ratiometric calibration.

Sodium current‐induced calcium signals in isolated guinea‐pig ventricular myocytes.

1. Na+ current (INa)‐induced Ca2+ transients were studied in ventricular myocytes isolated from adult guinea‐pig hearts. The fluorescent Ca2+ indicator fluo‐3 or a mixture of fluo‐3 and fura‐red were used in conjunction with confocal microscopy to follow the intracellular Ca2+ concentration while membrane currents were measured simultaneously with the whole‐cell configuration of the patch‐clamp technique. 2. Ca2+ release from the sarcoplasmic reticulum (SR) could be triggered either by Ca2+ current (ICa) or Na+ current (INa). Analysis of INa‐induced Ca2+ signals at higher temporal resolution revealed a faster upstroke of these transients when compared with those triggered by ICa. 3. In the presence of 20 microM ryanodine to block SR Ca2+ release ICa elicited a verapamil‐sensitive Ca2+ transient with a slow upstroke. INa also induced a residual Ca2+ transient that was insensitive to 10 microM verapamil and characterized by a rapid upstroke. 4. The existence of a residual Ca2+ transient in the absence of SR Ca2+ release and L‐type ICa indicates that INa is indeed able to evoke an increase in [Ca2+]i without uncontrolled activation of Ca2+ channels. 5. Substitution of extracellular Na+ by Li+ suppressed INa‐induced Ca2+ transients, suggesting that the Ca2+ release and the residual Ca2+ transient can only be elicited by influx of Na+ and not by Li+. This result supports the notion that both the residual Ca2+ transient as well as the INa‐induced Ca2+ release are mediated by the Na(+)‐Ca2+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Submicroscopic calcium signals as fundamental events of excitation‐‐contraction coupling in guinea‐pig cardiac myocytes.

1. Subcellularly localized Ca2+ signals have been proposed to represent elementary events of cardiac Ca2+ signalling (Ca2+ sparks), whereby an individual sarcolemmal L‐type Ca2+ channel locally controls opening of a single (or a few) Ca2+ release channels in the sarcoplasmic reticulum (SR). 2. To investigate directly the elementary nature of this Ca(2+)‐induced Ca2+ release mechanism we used flash photolysis of caged Ca2+ while simultaneously measuring the intracellular Ca2+ concentration ([Ca2+]i) with a laser‐scanning confocal microscope. 3. Power spectral analysis of the confocal images performed in the spatial domain revealed that only Ca2+ signalling events involving the L‐type Ca2+ channel pathway gave rise to Ca2+ sparks. In contrast, SR Ca2+ release triggered by photolytic [Ca2+]i jumps resulted in Ca2+ transients that were always spatially homogeneous. 4. From these findings we conclude that the fundamental event of Ca2+ signalling in cardiac muscle may be smaller in size or amplitude than a Ca2+ spark. 5. We term this event a ‘Ca2+ quark’ possibly resulting from gating of a single SR Ca2+ release channel. It is proposed that concerted activation of several ‘Ca2+ quarks’ may be required for a Ca2+ spark. The ‘Ca2+ quark’ could also be the fundamental event in other cell types implementing a hierarchical Ca2+ signalling concept.

Voltage-independent calcium release in heart muscle

The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2(+)-induced Ca2+ release.

Fundamental calcium release events revealed by two‐photon excitation photolysis of caged calcium in guinea‐pig cardiac myocytes

1 In cardiac muscle, ‘Ca2+ sparks’ have been proposed to underlie Ca2+‐induced Ca2+ release (CICR), and to result from openings of clusters of Ca2+ channels (ryanodine receptors; RyRs) located in the sarcoplasmic reticulum membrane. 2 To investigate the elementary nature of these Ca2+ signals directly, a diffraction‐limited point source of Ca2+ was created in single cardiac myocytes by two‐photon excitation photolysis of caged Ca2+. Simultaneously, concentration profiles of released Ca2+ were imaged at high temporal and spatial resolution with a laser‐scanning confocal microscope. 3 This approach enabled us to generate and detect photolytic Ca2+ signals that closely resembled the Ca2+ sparks occurring naturally, not only in amplitude and size, but also in their ability to trigger additional Ca2+ sparks or Ca2+ waves. 4 Surprisingly, at low photolytic power minuscule events with estimated Ca2+ release fluxes 20‐40 times smaller than those calculated for a typical Ca2+ spark were directly resolved. These events appeared to arise from the opening of a more limited number of RyRs (possibly one) or from RyRs exhibiting a different gating mode and may correspond to the elusive ‘Ca2+ quark’. 5 The Ca2+ quark represents the fundamental Ca2+ release event of excitable cells implementing hierarchical Ca2+ signalling systems with Ca2+ release events of various but distinct amplitude levels (i.e. Ca2+ quarks, Ca2+ sparks and full cellular Ca2+ transients). 6 A graded recruitment of nanoscopic Ca2+ release domains (i.e. Ca2+ quarks) exhibiting variable degrees of spatial coherence and coupling may then build up intermediate Ca2+ signalling events (i.e. Ca2+ sparks). This mechanism suggests the existence of Ca2+ sparks caused by gating of a variable fraction of RyRs from within an individual cluster. Additional mobilization of a variable number of these Ca2+ sparks enables cardiac cells to show graded cellular Ca2+ transients. Similar recruitment processes may underlie regulation of Ca2+ signalling on the cellular level in general.

Microscopic spiral waves reveal positive feedback in subcellular calcium signaling

The regenerative Ca(2+)-induced Ca2+ release mechanism is an important amplifier of signal transduction in diverse cells. In heart muscle cells, this mechanism contributes to the Ca2+ transient activating the mechanical contraction, but it is also believed to drive Ca2+ waves propagating within the cytosol. We investigated the subcellular Ca2+ distribution in heart muscle cells during spontaneous Ca2+ release using laser scanning confocal microscopy with a ratiometric fluorescent indicator technique. Besides planar Ca2+ waves with linear propagation, sequences of confocal optical sections also revealed spiral Ca2+ waves spinning around a subcellular core at approximately 1 Hz. Although the Ca2+ spirals were continuous processes they frequently exhibited an apparently oscillatory output function into the elongated cell body. These oscillatory waves emanating from the spiral at regular intervals were formally considered to be short outer segments of the spiral but could not be distinguished from planar Ca2+ waves propagating along the longitudinal cell axis. The complex spatiotemporal pattern of spiral Ca2+ waves implies the participation of an active process exhibiting a large degree of positive feedback, most likely the Ca(2+)-induced Ca2+ release mechanism.

The nitrodibenzofuran chromophore: A new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16–160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M−1 cm−1). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca2+ rapidly released Ca2+ (rate of 20,000 s−1) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of ∼0.6 GM and two-photon photolysis induced localized Ca2+-induced Ca2+ release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Modulation of Ca2+ release in cultured neonatal rat cardiac myocytes. Insight from subcellular release patterns revealed by confocal microscopy.

It is well established that in heart muscle the influx of Ca2+ through Ca2+ channels during the action potential is the main trigger for Ca2+ release from the sarcoplasmic reticulum (SR), but intact cardiac tissue and single myocytes are also known to exhibit spontaneous Ca2+ release from the SR under a variety of circumstances. Although conditions favoring spontaneous activity have been examined extensively, mechanisms modulating or regulating spontaneous as well as triggered Ca2+ release are still largely unknown. Using the high spatial and temporal resolution of laser-scanning confocal microscopy, we investigated subcellular aspects of spontaneous and triggered Ca2+ release in isolated rat neonatal myocytes loaded with the Ca(2+)-sensitive fluorescent dye fluo 3. Three distinct patterns of spontaneous Ca2+ release were identified: (1) a homogeneous Ca2+ release, presumably corresponding to Ca2+ release during a spontaneous action potential, (2) a focal or spatially restricted Ca2+ release with no or only limited subcellular propagation, and (3) a Ca2+ release propagating as a wave throughout the entire cell. Pharmacologic tools that interfere with the SR revealed that all release types were critically dependent on the Ca2+ release and uptake function of the SR. From our results we conclude that the probability, extent, and pattern of Ca2+ release are modulated on the subcellular level. The observed spectrum of release patterns can be explained by a space- and time-dependent variability in the positive feedback of the Ca(2+)-induced Ca(2+)-release mechanism within an individual myocyte. Presumably, this variability depends on the existence of subcellular functional elements of the SR. The actual degree of positive feedback may be modulated locally by the Ca(2+)-loading state of each SR element.

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

1 Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. 2 To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK‐N‐MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. 3 Expression of the EpoR in SK‐N‐MC cells was detected by reverse transcription‐PCR, Western blot and immunofluorescence analysis. 4 Patch‐clamp studies of SK‐N‐MC cells confirmed the expression of T‐type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. 5 Confocal laser scanning microscopy analysis of SK‐N‐MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. 6 The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T‐type Ca2+ channel blocker flunarizine. 7 This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T‐type voltage‐dependent Ca2+ channels.