Photorelease Of Ca2 Research Articles

Calcium Signaling in Muscle Cells from a Patient Cohort with Inheritable Muscle Diseases

Malignant Hyperthermia Susceptibility, MHS, is a hereditary sub-clinical condition characterized by events of uncontrolled release of calcium, deadly if untreated. 60% of MHS cases are due to mutations of the RyR1 Ca release channel. A puzzling property of these mutations is their phenotypic variability. We are comparing Ca signals in cells from MHS patients with those from normal donors. Every subject is genotyped for the proteins of interest. Gracilis muscle biopsies taken in Toronto from at risk patients, are received in Chicago overnight and studied acutely with 2-silicone gap voltage clamp, or used to generate cultures (technique by S Treves, Basel). Myotubes are imaged for [Ca2+]cyto (with fluo-4) and Vm (with di-8-ANEPPS). In response to brief field stimuli, cells from healthy subjects had normal action potentials (t ∼10 ms) and a monophasic Ca transient, rarely followed by a slow wave. Cells from a patient with the MH-linked mutation G341R always had a 2-phase response --a small initial transient followed by a large propagated wave. Cells from two MHS patients with normal genotype (who were positive for one of 2 standard MH tests) had responses with intermediate features. Using pharmacology and photorelease of Ca2+ and IP3, we are testing various mechanisms that could be altered, including gating control of RyRs as well as IP3 receptor channels, which contribute to slow transients in myotubes (e.g. Casas et al., 2010). Given the variety of phenotypes and genotypes available, these approaches should allow the characterization of cellular signaling defects in multiple mutations, linked to MH and other muscle diseases. The comparison should increase our understanding of disease mechanisms as well as our insight on structure-function relationships of the RyR channel. Funded by NIAMS/NIH and A.H.A.

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Ca2+ Pulses Control Local Cycles of Lamellipodia Retraction and Adhesion along the Front of Migrating Cells

Ca(2+) signals regulate polarization, speed, and turning of migrating cells. However, the molecular mechanism by which Ca(2+) acts on moving cells is not understood. Here we show that local Ca(2+) pulses along the front of migrating human endothelial cells trigger cycles of retraction of local lamellipodia and, concomitantly, strengthen local adhesion to the extracellular matrix. These Ca(2+) release pulses had small amplitudes and diameters and were triggered repetitively near the leading plasma membrane with only little coordination between different regions. We show that each Ca(2+) pulse triggers contraction of actin filaments by activating myosin light-chain kinase and myosin II behind the leading edge. The cyclic force generated by myosin II operates locally, causing a partial retraction of the nearby protruding lamellipodia membrane and a strengthening of paxillin-based focal adhesion within the same lamellipodia. Photo release of Ca(2+) demonstrated a direct role of Ca(2+) in triggering local retraction and adhesion. Together, our study suggests that spatial sensing, forward movement, turning, and chemotaxis are in part controlled by confined Ca(2+) pulses that promote local lamellipodia retraction and adhesion cycles along the leading edge of moving cells.

Defining the Role of Astrocytes in Neuromodulation

Astrocytes undergo elevations in intracellular calcium following activation of metabotropic receptors, which may trigger glutamate secretion and excitation of surrounding neurons. In this issue of Neuron, Fiacco et al. use transgenic mice that express a foreign G(q)-coupled receptor in astrocytes to show that selective stimulation of astrocytes is not sufficient to induce the release of glutamate.

On the Loose: Uncaging Ca2+-induced Ca2+ Release in Smooth Muscle

On the Loose: Uncaging Ca2+-induced Ca2+ Release in Smooth Muscle

Protein Kinase Activation Increases Insulin Secretion by Sensitizing the Secretory Machinery to Ca2+

Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca2+, we characterize three kinetically different pools of vesicles in rat pancreatic β-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is ∼20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca2+, we find that the HCSP is a distinct population of vesicles from those colocalized with Ca2+ channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca2+.

Cross-Bridge versus Thin Filament Contributions to the Level and Rate of Force Development in Cardiac Muscle

In striated muscle thin filament activation is initiated by Ca 2+ binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca 2+ activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment ( k tr) in demembranated rat cardiac trabeculae as [Ca 2+] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca 2+ activation by comparing the rate of force development ( k Ca) after a step increase in [Ca 2+] with photorelease of Ca 2+ from NP-EGTA to maximal k tr, where Ca 2+ binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca 2+ activation. At similar submaximal levels of steady-state force there was no increase in k tr with dATP, but k tr was enhanced at higher Ca 2+ concentrations, resulting in an extension (not elevation) of the k tr-force relationship. Interestingly, we found that maximal k tr was faster than k Ca, and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca 2+-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca 2+.

Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca 2+ release: a central role of inositol 1,4,5-trisphosphate receptors

The dynamics of carbachol (CCh)-induced [Ca 2+] i changes was related to the kinetics of muscarinic cationic current (mI cat) and the effect of Ca 2+ release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP 3Rs) on mI cat was evaluated by fast x– y or line-scan confocal imaging of [Ca 2+] i combined with simultaneous recording of mI cat under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca 2+-sensitive indicator fluo-3, x– y confocal imaging revealed CCh (10 μM)-induced Ca 2+ waves, which propagated from the cell ends towards the myocyte centre at 45.9±8.8 μm s −1 ( n=13). Initiation of the Ca 2+ wave preceded the appearance of any measurable mI cat by 229±55 ms ( n=7). Furthermore, CCh-induced [Ca 2+] i transients peaked 1.22±0.11 s ( n=17) before mI cat reached peak amplitude. At −50 mV, spontaneous release of Ca 2+ through RyRs, resulting in Ca 2+ sparks, had no effect on CCh-induced mI cat but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca 2+ release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI cat ( n=14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI cat when [Ca 2+] i was strongly buffered with Ca 2+/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca 2+ sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca 2+ upon cell dialysis with 5 mM NP–EGTA/3.8 mM Ca 2+ potentiated mI cat in the same way as in control). In contrast, IP 3R-mediated Ca 2+ release upon flash photolysis of “caged” IP 3 (30 μM in the pipette solution) augmented mI cat ( n=15), even though [Ca 2+] i did not reach the level required for potentiation of mI cat during photorelease of Ca 2+ ( n=10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca 2+ indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP 3R type 1 and to RyRs revealed that IP 3Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP 3R-mediated Ca 2+ release plays a central role in the modulation of mI cat in the guinea-pig ileum and that IP 3 may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca 2+.

Molecular Basis for Pacemaker Cells in Epithelia

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.

The effects of changing intracellular Ca 2+ buffering on the excitability of cultured dorsal root ganglion neurones

The whole cell patch clamp technique was used to study the effects of changes in [Ca 2+] i on the excitability of cultured dorsal root ganglion (DRG) neurones. Increases in [Ca 2+] i caused membrane depolarization, altered the characteristics of evoked action potentials and activated potassium, chloride and non-selective cation conductances. Mobilization of Ca 2+ from intracellular stores with caffeine (1 mM) or ryanodine (10 μM) or photorelease of Ca 2+ from DM-nitrophen gave rise to depolarizations suggesting a dominant inward current under our recording conditions of low [Ca 2+] i buffering capacity. The actions of [Ca 2+] i could be partially reversed by intracellular flash photolysis of diazo-2 but not by diazo-3. The electrophysiological properties of some DRG neurones were not influenced by changes in [Ca 2+] i and we suggest that this is because in this heterogeneous culture some neurones do not express Ca 2+-activated ion channels.

In vivo regulation of axon extension and pathfinding by growth-cone calcium transients.

Growth cones at the tips of extending neurites migrate through complex environments in the developing nervous system and guide axons to appropriate target regions using local cues. The intracellular calcium concentration ([Ca2+]i) of growth cones correlates with motility in vitro, but the physiological links between environmental cues and axon growth in vivo are unknown. Here we report that growth cones generate transient elevations of [Ca2+]i as they migrate within the embryonic spinal cord and that the rate of axon outgrowth is inversely proportional to the frequency of transients. Suppressing Ca2+ transients by photorelease of a Ca2+ chelator accelerates axon extension, whereas mimicking transients with photorelease of Ca2+ slows otherwise rapid axonal growth. The frequency of Ca2+ transients is cell-type specific and depends on the position of growth cones along their pathway. Furthermore, growth-cone stalling and axon retraction, which are two important aspects of pathfinding, are associated with high frequencies of Ca2+ transients. Our results indicate that environmentally regulated growth-cone Ca2+ transients control axon growth in the developing spinal cord.

Two-photon and UV-laser flash photolysis of the Ca2+cage, dimethoxynitrophenyl-EGTA-4

We report efficient two-photon and UV-laser flash photolysis of dimethoxynitrophenyl-EGTA-4 (DMNPE-4), a newly-developed photolabile Ca2+-specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg2+affinity (≈7 mM), a Kdfor Ca2+of 19 nM, a quantum yield of 0.20 and changes its Ca2+affinity by 21 000-fold upon photolysis. Two-photon excitation photolysis (TPP) experiments were performed with a Ti:Sapphire laser in solutions containing DMNPE-4 with either 0 or 10 mM Mg2+and compared to that of the widely used Ca2+cage, DM-nitrophen (Kdfor Ca2+5 nM, Kdfor Mg2+2.5 μM, quantum yield 0.18, affinity change 600 000-fold). The resulting Ca2+signals were recorded with the fluorescent Ca2+indicator fluo-3 and a laser-scanning confocal microscope in the line-scan mode. In vitro, photolysis of DMNPE-4:Ca2+produced Ca2+-release signals that had comparable amplitudes and time courses in the presence and absence of Mg2+. However, photorelease of Ca2+from DM-nitrophen was obviated by the presence of Mg2+. In patch-clamped isolated cardiac myocytes, equivalent TPP results were obtained in analogous experiments. Single-photon excitation of DMNPE-4 by Nd:YAG laser flashes produced Na–Ca exchange currents of comparable amplitude in the absence and presence of Mg2+. However, only very small currents were observed in DM-nitrophen solution containing 10 mM Mg2+. In conclusion, both DMNPE-4 and DM-nitrophen undergo TPP, however, only DMNPE-4 exhibits efficient release of Ca2+in the presence of Mg2+.

Determinants of relaxation rate in skinned frog skeletal muscle fibers.

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10 degreesC. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+ concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.

Endocytosis of secretory granules in mouse pancreatic beta‐cells evoked by transient elevation of cytosolic calcium.

1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta-cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50-100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo- and endocytosis was studied by photorelease of Ca2+ from the 'caged' precursor Ca(2+)-nitrophenyl-EGTA (Ca(2+)-NP-EGTA). Both exo- and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg-ATP. 7. Our data indicate that in the pancreatic beta-cell, exocytosis and endocytosis are regulated by different mechanisms.

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.

Electrophysiological and ultrastructural events evoked by methacholine and intracellular photolysis of caged compounds in cultured ovine trachea submucosal gland cells

Cultured ovine trachea submucosal gland cells release lysozyme in response to extracellular application of secretagogues, including the muscarinic receptor agonist methacholine (20 microM). Investigation of the ultrastructure has shown that these cells contain electron-dense cored granules, which differ from the intact tissue, but appear to be released in response to the application of methacholine and can be arrested during exocytosis by the application of tannic acid. The release process appears to be linked to electrophysiological events activated by methacholine. Extracellular application of methacholine and intracellular photorelease of Ca2+ from DM-nitrophen evoked similar events suggesting that a rise in intracellular Ca2+ may occur following muscarinic receptor activation. Measurements of the reversal potential and the inhibitory action of the chloride channel blocker niflumic acid (10 microM) indicated that Ca(2+)-activated Cl- channel activity underlies these events. Some of the cultured submucosal gland cells also responded similarly to intracellular photorelease of inositol 1,4,5-trisphosphate, suggesting a possible link between muscarinic receptor occupation by agonist, release of calcium from stores, and activation of Ca(2+)-activated Cl- current. Secretion of lysozyme, methacholine-activated currents and currents evoked by intracellular photorelease of Ca2+ were also attenuated by the potent bronchodilator Ro 31-6930 (1 microM). We conclude that Ca(2+)-activated Cl- conductances play an important role in secretory processes in cultured submucosal gland cells. This may have a bearing on both physiological control of secretory events and regulation of the nature of airway surface liquid. Ca(2+)-activated Cl- channels may offer a potential target site for novel therapeutic agents.

Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation.

Channel adaptation is a fundamental feature of sarcoplasmic reticulum calcium release channels (called ryanodine receptors, RyRs). It permits successive increases in the intracellular concentration of calcium (Ca2+) to repeatedly but transiently activate channels. Adaptation of RyRs in the absence of magnesium (Mg2+) and adenosine triphosphate is an extremely slow process (taking seconds). Photorelease of Ca2+ from nitrophenyl-EGTA, a photolabile Ca2+ chelator, demonstrated that RyR adaptation is rapid (milliseconds) in canine heart muscle when physiological Mg2+ concentrations are present. Phosphorylation of the RyR by protein kinase A increased the responsiveness of the channel to Ca2+ and accelerated the kinetics of adaptation. These properties of the RyR from heart may also be relevant to other cells in which multiple agonist-dependent triggering events regulate cellular functions.

Kinetics of tension development in skinned cardiac myocytes measured by photorelease of Ca2+

The rate of activation of tension development by free Ca2+ was examined in skinned rat ventricular myocytes. Pulse photolysis of the photosensitive Ca2+ chelator, Nitr-7, was used to rapidly elevate Ca2+ in the vicinity of the myofilaments. Tension increased exponentially with a first-order rate constant (kCa) that depended on the level of Ca2+ activation. kCa increased approximately linearly from 0.9 +/- 0.2 s-1 at 20% maximal Ca(2+)-activated tension (Po) to 4.0 +/- 0.9 s-1 at 85% Po, representing a fourfold increase in kCa with activation. Reducing free Mg2+ from 1 to 0.1 mM accelerated kCa by about twofold at all levels of Ca2+. Tension development kinetics were significantly different in skinned rabbit psoas fibers: kCa increased nonlinearly from 1.2 +/- 0.2 s-1 at 15% Po to a maximum of 17.5 +/- 1.3 s-1 at 85% Po, representing a 15-fold increase in kCa with activation. Moreover, lowering free Mg2+ increased kCa only at submaximal Ca2+ in psoas fibers. Extraction of troponin C (TNC) from psoas fibers and recombination with bovine cardiac TNC did not significantly alter any of these characteristics of tension development kinetics. We conclude that significant differences exist between cardiac and fast-twitch skeletal muscles in terms of the effects of Ca2+ and Mg2+ on contraction kinetics and that these differences cannot be attributed solely to differences in TNC isoforms.

Confocal microfluorimetry of Ca2+ signals evoked in Xenopus oocytes by photoreleased inositol trisphosphate.

1. The subcellular characteristics of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ liberation were studied in Xenopus oocytes by the use of confocal microfluorimetry to monitor Ca2+ signals from minutely localized region of the cell in response to photorelease of InsP3 from a caged precursor. 2. Photorelease of increasing amounts of InsP3 by progressively longer light flashes evoked transient Ca2+ responses that appeared abruptly at a certain threshold duration, and then grew steeply over a narrow range of flash durations to reach a maximum. Further lengthening of flash duration gave no increase in size of the Ca2+ signals, but their rate of rise continued to increase and their duration became longer. Simultaneous measurements of Ca(2+)-activated Cl- currents showed a slightly higher threshold than the Ca2+ signal, and a more graded dependence upon flash duration. 3. The threshold flash durations required to evoke Ca2+ and membrane current signals grew by more than 100-fold as the area of the oocyte exposed to photolysis light was reduced from a square of 140 microns to 5 microns. 4. Ca2+ signals evoked by photoreleased InsP3 began following a dose-dependent latency that was as long as several seconds with low intensity light, but shortened to about 50 ms at maximum intensity. The extrapolated minimum latency with infinite photorelease of InsP3 was about 30 ms. 5. InsP3-evoked membrane currents began 30 ms or longer after the corresponding Ca2+ signals, whereas currents evoked by photorelease of Ca2+ from a caged precursor began within 5 ms of the onset of the light flash. 6. No differences in duration of InsP3-evoked Ca2+ signals were apparent when the confocal measuring spot was positioned close to the plasma membrane or about 10 microns more deeply into the oocyte. At both locations the Ca2+ signals were more prolonged than the associated membrane current signals. 7. Ca2+ signals to a test light flash were suppressed for about 2 s following a conditioning suprathreshold flash, but recovered almost completely after 6 s. The associated membrane current signals were facilitated at short intervals, suppressed at intervals between 0.5 and 3 s, and subsequently recovered more slowly than the Ca2+ signals. 8. Photorelease of InsP3 during 30 s exposures of low intensity evoked trains of repetitive Ca2+ spikes. The overall amplitudes of these responses changed little with increasing in frequency, and became smaller and superimposed on a more sustained elevation of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)