Biomedical subjects
Publications and source records attributed to P Lipp.
The 'bell-shaped' curve relating cytosolic Ca(2+) concentration to IP(3) receptor activation is considered important in the generation of the complex Ca(2+) signals seen inside many cells. But recent findings suggest this bimodal relationship is not always evident and may not apply to some IP(3) receptor isoforms.
Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.
We report the sequence, structure and distribution of a novel transient receptor potential (trp) homologue from Xenopus, Xtrp, determined by screening an oocyte cDNA library. On the basis of sequence similarity and predicted structure, Xtrp appears to be a homologue of mammalian trp1 proteins. Two polyclonal antibodies raised against distinct regions of the Xtrp sequence revealed Xtrp expression in various Xenopus tissues, and the localization of Xtrp at the plasma membrane of Xenopus oocytes and HeLa cells. Since capacitative calcium entry into Xenopus oocytes has been shown previously to be substantially inhibited by trp1 antisense oligonucleotides [Tomita, Kaneko, Funayama, Kondo, Satoh and Akaike (1998) Neurosci. Lett. 248, 195-198] we suggest that Xtrp may underlie capacitative calcium entry in Xenopus tissues.
Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.
Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.
The generation of oscillations and global Ca2+ waves relies on the spatio-temporal recruitment of elementary Ca2+ signals, such as 'Ca2+ puffs'. Each elementary signal contributes a small amount of Ca2+ into the cytoplasm, progressively promoting neighboring Ca2+ release sites into an excitable state. Previous studies have indicated that increases in frequency or amplitude of such hormone-evoked elementary Ca2+ signals are necessary to initiate Ca2+ wave propagation. In the present study, an electroporation device was used to rapidly and reversibly permeabilize the plasma membrane of HeLa cells and to allow a limited influx of Ca2+. With low field intensities (100-500 V/cm), brief (50-100 micros) electroporation triggered localized Ca2+ signals that resembled hormone-evoked Ca2+ puffs, but not global signals. With such low intensity electroporative pulses, the Ca2+ influx component was usually undetectable, confirming that the electroporation-induced local signals represented Ca2+ puffs arising from the opening of intracellular Ca2+ release channels. Increasing either the frequency at which low-intensity electroporative pulses were applied, or the intensity of a single electroporative pulse (>500 V/cm), resulted in caffeine-sensitive regenerative Ca2+ waves. We suggest that Ca2+ puffs caused by electroporation functionally mimic hormone-evoked elementary events and can activate global Ca2+ signals if they provide a sufficient trigger.
Previous studies of (InsP3)-evoked elementary Ca2+ events suggested a hierarchy of signals; fundamental events ("Ca2+ blips") arising from single InsP3 receptors (InsP3Rs), and intermediate events ("Ca2+ puffs") reflecting the coordinated opening of a cluster of InsP3Rs. The characteristics of such elementary Ca2+ release signals provide insights into the functional interaction and distribution of InsP3Rs in living cells. Therefore we investigated whether elementary Ca2+ signaling is truly represented by such stereotypic release events. A histogram of >900 events revealed a wide spread of signal amplitudes (20-600 nM; mean 216 +/- 4 nM; n = 206 cells), which cannot be explained by stochastic variation of a stereotypic Ca2+ release site. We identified elementary Ca2+ release sites with consistent amplitudes (<20% difference) and locations with variable amplitudes (approximately 500% difference). Importantly, within single cells, distinct sites displayed events with significantly different mean amplitudes. Additional determinants affecting the magnitude of elementary Ca2+ release were identified to be (i) hormone concentration, (ii) day-to-day variability, and (iii) a progressively decreasing Ca2+ release during prolonged stimulation. We therefore suggest that elementary Ca2+ events are not stereotypic, instead a continuum of signals can be achieved by either recruitment of entire clusters with different numbers of InsP3Rs or by a graded recruitment of InsP3Rs within a cluster.
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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.
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It is known that the nucleoplasmic ionised calcium concentration (Can) controls nuclear functions such as transcription, although the source and nature of the signals which modulate Can are unclear. Using confocal imaging, we investigated the subcellular origin of Can signals in Fluo-3-loaded HeLa cells. Our data indicate that all signals which increased Can were of cytoplasmic origin. Can was elevated during the propagation of global Ca waves within cells. More strikingly, we found that individual cytoplasmic elementary release events e.g. Ca puffs, evoked by physiological levels of stimulation, caused transient Can increases. Significantly, >70% of all Ca puffs originated within a 2-3 micron perinuclear zone and propagated anisotropically across the entire nucleus. Due to the relatively slow relaxation of Can transients compared with those in the cytoplasm, repetitive perinuclear Ca puffs were integrated into a 'staircase' of increasing Can. Due to the effective diffusion of Ca in the nucleoplasm, the nucleus served as a 'Ca tunnel', distributing Ca to parts of the cytosol which were otherwise not within the cytoplasmic diffusion radii of Ca puffs. Given the close proximity of the majority of puff sites to the nucleus, it seems that the elementary Ca release system is designed to facilitate nuclear Ca signalling. Consequently, Ca-dependent regulation of nuclear function must be considered at the microscopic elementary level.
Recent studies have suggested that global intracellular Ca2+ signals arise from the summation and coordination of subcellular elementary release events (e.g., "Ca2+ puffs"), although the modes of recruitment of such signals are unknown. In order to understand how cells utilize elementary Ca2+ release events, we imaged Ca2+ transients evoked through the phosphoinositide pathway in HeLa cells using confocal microscopy. During the pacemaker phase leading to the global Ca2+ signal, elementary Ca2+ release events were recruited in (1) frequency, (2) amplitude, and (3) spatial domains. Since each digital elementary event contributes to a small change of the analog cytosolic Ca2+ concentration, the net effect of the advancement in the three domains is to drive the ambient Ca2+ concentration toward a threshold where the signal becomes regenerative, resulting in a global Ca2+ wave.
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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.
1. Ratiometric confocal microscopy and the whole-cell patch clamp technique were used to simultaneously record intracellular Ca2+ transients and membrane currents from guinea-pig ventricular myocytes. Intracellular dialysis with the low-affinity Ca2+ buffer citrate enabled us to record and analyse Ca2+ transients caused by Ca2+ influx alone and by additional Ca2+ release from the sarcoplasmic reticulum (SR) in the same cell. 2. In freshly isolated adult myocytes (used within 1-4 h of isolation) both types of Ca2+ transients ('Ca2+ entry' and 'Ca2+ release' transients) were spatially uniform regardless of the Ca2+ current (ICa) duration. In contrast, Ca2+ transients in short-term cultured (1-2 days) myocytes exhibited marked spatial inhomogeneities. ICa frequently evoked Ca2+ waves that propagated from either or both ends of the cardiac myocyte. Reduction of the ICa duration caused Ca2+ release that was restricted to one of the two halves of the cell. 3. Analysis of the Ca2+ entry signals in freshly isolated and short-term cultured myocytes indicated that the spatial properties of the Ca2+ influx signal were responsible for the spatial properties of the triggered Ca2+ release from the SR. In freshly isolated ventricular myocytes Ca2+ influx was homogeneous while in short-term cultured cells pronounced Ca2+ gradients could be found during Ca2+ influx. Spatial non-uniformities in the amplitude of local Ca2+ entry transients were likely to cause subcellularly restricted Ca2+ release. 4. The changes in the spatial properties of depolarization-induced Cai2+ signals during short-term culture were paralleled by a decrease (to 65%) in the total cell capacitance. In addition, staining the sarcolemma with the membrane-selective dye Di-8-ANEPPS revealed that, in cultured myocytes, t-tubular membrane connected functionally to the surface membrane was reduced or absent. 5. These results demonstrate that the short-term culture of adult ventricular myocytes results in the concomitant loss of functionally connected t-tubular membrane. The lack of the t-tubular system subsequently caused spatially non-uniform SR Ca2+ release. Evidence is presented to show that in ventricular myocytes lacking t-tubules non-uniform SR Ca2+ release was, most probably, the result of inhomogeneous Ca2+ entry during ICa. These findings directly demonstrate the functional importance of the t-tubular network for uniform ventricular Ca2+ signalling.
1. Spatiotemporal aspects of subcellular Ca2+ signalling were studied in cultured adult guinea-pig atrial myocytes. A mixture of the Ca2+ indicators fluo-3 and Fura Red in combination with laser-scanning confocal microscopy was used for [Ca2+]i measurements while membrane currents were recorded simultaneously. 2. In citrate-loaded atrial myocytes not every Ca2+ current (ICa) could trigger Ca2+ release from the sarcoplasmic reticulum (SR). Two types of Ca2+ signals could be observed: Ca2+ transients resulting from (i) Ca2+ influx alone and (ii) additional Ca2+ release. 3. Ca2+ release elicited by voltage steps of 100-150 ms duration was either apparently homogeneous or propagated as Ca2+ waves through the entire cell. With brief ICa (50-75 ms), Ca2+ waves with limited subcellular propagation were observed frequently. These waves always originated from either end of the myocyte. 4. The time course of changes in Na(+)-Ca2+ exchange current (INaCa) depended on the subcellular properties of the underlying Ca2+ transient and on the particular cell geometry. Apparently homogeneous Ca2+ release was accompanied by an inward change of INaCa the onset phase of which was fused with ICa. Changes in INaCa caused by a Ca2+ wave propagating through the entire cell showed a W shape, which could be attributed to differences of the fractional surface-to-volume ratio in different cell segments during propagation of the Ca2+ wavefront. Those waves with limited spreading only activated a small component of INaCa. 5. The different subcellular patterns of Ca2+ release signals can be explained by spatial inhomogeneities in the positive feedback of the SR. This depends on the local SR Ca2+ loading state under the control of the local Ca2+ influx during activation of ICa. Due to the higher surface-to-volume ratio at the two ends of the myocyte, SR loading and therefore the positive feedback in Ca(2+)-induced Ca2+ release may be higher at the ends, locations where Ca2+ waves are preferentially triggered. 6. We conclude that the individual cell geometry may be an important determinant of subcellular Ca2+ signalling not only in cardiac muscle cells but presumably also in other types of cells that depend on Ca2+ signalling. In addition, the cell geometry in combination with varying subcellular Ca2+ release patterns can greatly affect the time course of Ca(2+)-activated membrane currents.
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