Photorelease Of Ca2 Research Articles

Mechanics and structure of cross-bridges during contractions initiated by photolysis of caged Ca2+.

Cross-bridge structure and mechanics were studied during development of skinned frog muscle fiber contractions initiated by photolysis of DM-nitrophen (a caged Ca2+). Stiffness rises earlier than tension following photo-release of Ca2+. A similar lead of stiffness in electrically stimulated fibers and the early rise of the I11/I10 ratio of equatorial X-ray reflections are thought to signal attachment of cross-bridges into states with lower force than in steady-state contraction. We investigated the structure of the early attachments by electron microscopy of fibers activated by photolysis of DM-nitrophen and then ultra-rapidly frozen and freeze substituted with tannic acid and OsO4. Sections from relaxed fibers show helical tracks of myosin heads on the thick filaments surface. Optical diffraction patterns show strong meridional intensities and layer lines up to the 6th order of 1/43 nm, indicating preservation and resolution of periodic structures smaller than 10 nm. Following photo-release of Ca2+, the 1/43 nm myosin layer line becomes less intense, and higher orders disappear. A approximately 1/36 nm layer line appears early (12-15 ms) and becomes stronger at later times. The 1/14.3 nm meridional spot weakens initially and recovers at a later time, while it broadens laterally. The 1/43 nm meridional spot is present during contraction, but the 2nd order meridional spot (1/21.5 nm) is weak or absent. These results are consistent with time resolved X-ray diffraction data on the periodic structures within the fiber. In sections along the 1,1 plane of activated fibers, the individual cross-bridges have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z-line. Fibers frozen at 13 ms, 33 ms, and 220 ms after photolysis all show surprisingly similar cross-bridges. Thus, a highly variable distribution of cross-bridge shapes and angles is established early in contraction.

Characteristics of membrane currents evoked by photoreleased inositol trisphosphate in Xenopus oocytes.

Photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor was used to study characteristics of Ca(2+)-activated Cl- currents activated in Xenopus oocytes by the InsP3-Ca2+ signaling pathway. Photolysis flashes shorter than a threshold duration evoked no response, but the current amplitude then grew about linearly as the flash duration was further lengthened. Currents directly evoked by photorelease of Ca2+ from a caged precursor grew linearly with increasing flash duration and showed a small threshold before they were activated. However, the major part of the threshold of InsP3-evoked responses appears to arise because a certain concentration of InsP3 (estimated to be approximately 60 nM) is required to evoke Ca2+ liberation. Subthreshold conditioning flashes potentiated responses to subsequent flashes, and the potentiation increased linearly with increasing conditioning flash duration before abruptly declining. The potentiation decayed exponentially with a time constant of approximately 17 s with increasing interflash interval. Currents evoked by photoreleased InsP3 began after a latency that shortened from 10 s or longer to 100 ms as the photolysis intensity was increased. This dose dependence of the latency could be quantitatively explained by the time required for the InsP3 concentration to rise above threshold. Intracellular injection of heparin (a competitive antagonist at the InsP3 receptor) increased the threshold for InsP3 action, as did increased temperature. We conclude that several characteristics of InsP3-evoked responses, including their dose dependence, latency, and facilitation with paired stimuli, arise because a distinct threshold level of InsP3 is required to evoke release of Ca2+ from intracellular stores.

Ca-induced Ca release from the SR of barnacle myofibrillar bundles, triggered by the photorelease of calcium ions from caged calcium.

We have triggered Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) of isolated myofibrillar bundles of barnacle striated muscle fibres using the rapid photorelease of Ca2+ from the caged calcium molecule, nitr-5 (Adams et al, 1988), thus overcoming diffusional delays normally inherent in such experiments.

Photorelease of Ca2+ Produces Na‐Ca Exchange Currents and Na‐Ca Exchange “Gating” Currentsa

Photorelease of Ca²⁺ Produces Na‐Ca Exchange Currents and Na‐Ca Exchange “Gating” Currents^a

Rapid activation by photolysis of nitr-5 in skinned fibres of the striated adductor muscle from the scallop

Photolysis of nitr-5, a caged calcium molecule, has been used for rapid activation of skinned fibre bundles of a myosin-regulated muscle, the striated adductor of the scallop, Pecten maximus. Chemically skinned fibre bundles (diameter 70–200 μm) were equilibrated in solutions containing 1–3 mM nitr-5 (p Ca 6.1) and then activated by ultraviolet laser pulse (25 ns). Pulse energies of 60–95 mJ gave contractions of over 90% maximum tension and a mean half-time for tension rise of 43 ms ( n = 4) at 12°C. Electrically stimulated bundles of intact fibres develop a tetanus with a rise half-time of 60.2 ms at 10°C ( n = 5) (Rall, J.A. (1981) J. Physiol. 321, 287–295, and personal communication). At lower pulse energies the skinned fibres gave smaller amplitude contractions with slower rates of rise (up to 260 ms half-time). In addition, a slower component of tension development (mean rise half-time 13.3 s) was often observed. In ATP-free solutions containing hexokinase and glucose, rigour tension developed with a delayed onset. Rapid release of ATP (0.47–0.59 mM) from photolysis of caged ATP (2 mM) at p Ca 4.5 then caused a rapid contraction with a mean half-time for tension development of 17 ms ( n = 4). The fast activation rates obtained by the photorelease of Ca 2+ from nitr-5 are similar to those obtained with skinned skeletal fibres of actin-regulated muscle. The results imply that the rate-limiting step in excitation-contraction coupling of the scallop muscle is not the increase in sarcoplasmic Ca 2+, but rather the activation of the muscle in response to this increase. The half-times of ATP-induced contractions at p Ca 4.5 suggest that in a contraction activated by a rapid Ca 2+ jump the process comprising ATP hydrolysis and cross-bridge cycling occurs at a somewhat faster rate than the Ca 2+-dependent activation process which preceds it.

Ca2(+)-induced Ca2+ release as examined by photolysis of caged Ca2+ in single ventricular myocytes

In cardiac muscle, entry of Ca2+ through the voltage-gated Ca2+ channel and its interaction with an intracellular site are thought to trigger the release of the intracellular Ca2+ pools and to activate contraction. The availability of a novel "caged calcium" compound, and its effective use in neuronal and heart cells to modulate Ca2+ channel and contraction, made it possible to examine directly the Ca2(+)-induced Ca2+ release hypothesis in intact mammalian cardiac myocytes. We used the caged Ca2+ compound DM-nitrophen, which on photolysis, rapidly (less than 200 microseconds) changes its Ca2(+)-binding affinity from 3 X 10(-9) to 2 X 10(-3) M at pH 7.0. In isolated whole cell clamped guinea pig ventricular myocytes dialyzed with unphotolyzed DM-nitrophen (Ca2+ buffered to values less than 10(-7) M), we found that a 160-microseconds light pulse photoreleased sufficient Ca2+ to activate contraction. Photorelease of Ca2+ failed to activate significant contraction in myocytes pretreated with caffeine, supporting the idea that the release of Ca2+ from intracellular pools was necessary to generate tension. However, photorelease of Ca2+ after the depolarization-induced Ca2+ release failed to suppress contraction, as predicted from the Ca2(+)-induced inactivation hypothesis. The failure to suppress contraction was not sufficient to definitively reject the Ca2(+)-induced inactivation hypothesis, since the intracellular Ca2+ concentration may not have risen sufficiently to inactivate the release channel.

Modulation of Ca2+ channel selectivity and cardiac contraction by photorelease of Ca2+

The effect of rapid increases of extracellular Ca2+ concentration on tension development and Ca2+ channel selectivity has been investigated in frog (Rana pipiens) ventricular myocardium using a novel light-sensitive Ca2+ chelator, dimethoxy-nitrophen. Dimethoxy-nitrophen is a photolabile tetracarboxylate Ca2+ chelator that on photolysis to dicarboxylate fragments alters its affinity (Kd) for Ca2+ from 5 X 10(-9) to 2 X 10(-3) M. A single 160-microseconds ultraviolet light pulse induced the release of approximately 80-100 microM Ca2+ with a half-time of 200 microseconds. In low extracellular Ca2+ concentration, Na+ current through the Ca2+ channel was blocked by photorelease of extracellular Ca2+ in less than 500 microseconds; nevertheless, 60-80 ms were required to activate or enhance tension. Enhancement of tension was more effective when Ca2+ was released during the activation of the Ca2+ channel than during its inactivation. The voltage dependence of enhanced tension, caused by photorelease of Ca2+, was bell shaped and was similar to that of Ca2+ current. These findings suggest that Ca2+ transport through the Ca2+ channel is the primary mechanism for the transport of Ca2+ to activate tension in the frog heart. The use of dimethoxy-nitrophen makes it possible to examine the rapid kinetics of the Ca-dependent processes involved in regulation of channel function and contraction.