Activation of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP.

The kinetics of force production in chemically skinned trabeculae from the guinea pig were studied by laser photolysis of caged ATP in the presence of Ca2+. Preincubation of the tissue during rigor with the enzyme apyrase was used to reduce the population of MgADP-bound cross-bridges (Martin and Barsotti, 1994). In untreated tissue, tension remained constant or dipped slightly below the rigor level immediately after ATP release, before increasing to the maximum measured in pCa 4.5 and 5 mM MgATP. The in-phase component stiffness, which is a measure of cross-bridge attachment, exhibited a large decrease before increasing to 55% of that measured in rigor. Neither the rate of the decline nor of the rise in tension was sensitive to the concentration of photolytically released ATP. The rate of the decline in stiffness was found to be dependent on [ATP]: 1.8 x 10(4) M-1/s-1, a value more than four times higher than that previously measured in similar experiments in the absence of Ca2+. The rate of tension development averaged 14.9 +/- 2.5 s-1. Preincubation with apyrase altered the mechanical characteristics of the early phase of the contraction. The rate and amplitude of the initial drop in both tension and stiffness after caged ATP photolysis increased and became dependent on [ATP]. The second-order rate constants measured for the initial drop in tension and stiffness were 8.4 x 10(4) M-1 s-1 and 1.5 x 10(5) M-1 s-1. These rates are more than two times faster than those previously measured in the absence of Ca2+. The effects of apyrase incubation on the time course of tension and stiffness were consistent with the hypothesis that during rigor, skinned trabeculae retain a significant population of MgADP-bound cross-bridges. These in turn act to attenuate the initial drop in tension after caged ATP photolysis and slow the apparent rate of rigor cross-bridge detachment. The results also show that Ca2+ increases the rate of cross-bridge detachment in both untreated and apyrase-treated tissue, but the effect is larger in untreated tissue. This suggests that in cardiac muscle Ca2+ modulates the rate of cross-bridge detachment.     

Kinetics of cardiac thin-filament activation probed by fluorescence polarization of rhodamine-labeled troponin C in skinned guinea pig trabeculae

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.     

Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.     

Kinetics of ATP hydrolysis and tension production in skinned cardiac muscle of the guinea pig

The kinetics of ATP hydrolysis and tension responses were studied simultaneously in a permeabilized preparation of cardiac tissue of the guinea pig. This was achieved by combining laserflash photolysis of P3-1-(2-nitrophenyl)ethyladenosine 5'-triphosphate ("caged-ATP") and a rapid freezing technique. In the presence of calcium ions, tension increased following the photolytic production of ATP with a half-time of 0.3 s. The timecourse of ATP hydrolysis consisted of an initial rapid phase followed by a steady-state hydrolysis rate of 0.4 s-1, indicating that the rate-limiting step of the ATPase in isometric fibers is slower and subsequent to the nucleotide hydrolysis step: the isometric steady state intermediate is probably an actomyosin-ADP complex. In the absence of calcium ions, rigor tension decreased upon the photolytic production of ATP with a half-time of 0.45 s. The time course of ATP hydrolysis was biphasic with a rapid initial phase of ATP hydrolysis, followed by a steady-state hydrolysis rate which was too slow to measure over the time scale of these experiments (less than 0.04 s-1). A comparison of the results obtained in this study with those reported for rabbit skeletal muscle reveals qualitative similarities between cardiac and skeletal muscle and also quantitative differences in their physiological and kinetic behavior.     

Activation kinetics of skinned cardiac muscle by laser photolysis of nitrophenyl-EGTA

The kinetics of Ca(2+)-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca(2+) released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca(2+)]. The time courses of the rise in stiffness and tension were biexponential at 23 degrees C, pH 7.1, and 200 mM ionic strength. At full activation (pCa < 5.0), the rates of the rapid phase of the stiffness and tension rise were 56 +/- 7 s(-1) (n = 7) and 48 +/- 6 s(-1) (n = 11) while the amplitudes were 21 +/- 2 and 23 +/- 3%, respectively. These rates had similar dependencies on final [Ca(2+)] achieved by photolysis: 43 and 50 s(-1) per pCa unit, respectively, over a range of [Ca(2+)] producing from 15% to 90% of maximal isometric tension. At all [Ca(2+)], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1 +/- 0.8 and 6.2 +/- 1.0 s(-1). The rate of this slower phase exhibited significantly less Ca(2+) sensitivity, 1 and 4 s(-1) per pCa unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca(2+)], suggest a mechanism of regulation in which Ca(2+) controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca(2+)] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.     

Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP.

We have measured the microsecond rotational motions of the sarcoplasmic reticulum (SR) Ca-ATPase as a function of enzyme-specific ligands, including those that induce active calcium transport. We labeled the Ca-ATPase with a maleimide spin probe and detected rotational dynamics using saturation-transfer electron paramagnetic resonance (ST-EPR). This probe's ST-EPR spectra have been shown to be sensitive to microsecond protein rotational motion, corresponding to large-scale protein rotations that should be affected by changes in the enzyme's shape, flexibility, protein-protein interactions (oligomeric state), and protein-lipid interactions. We found that the motions of the enzyme-nucleotide and the enzyme-nucleotide/Ca states are indistinguishable from the motions in the absence of ligands. Rotational mobility does decrease in response to the addition of DMSO, a solvent that inhibits Ca-ATPase activity and stabilizes the phosphoenzyme. However, the addition of phosphate to form phosphoenzyme, in the presence or absence of DMSO, does not change the motions significantly. During the steady state of active calcium transport, the microsecond rotational mobility is indistinguishable from that of the resting enzyme. In order to detect any transient changes in mobility that might not be detectable in the steady state and to improve the precision of steady-state measurements, we photolyzed caged ATP with a laser pulse in the presence of calcium and detected the ST-EPR response from the spin-labeled enzyme, with a time resolution of 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

Effects of magnesium pyrophosphate on mechanical properties of skinned smooth muscle from the guinea pig taenia coli.

Effects of the non-hydrolyzable nucleotide analogue magnesium pyrophosphate (MgPPi) on cross-bridge properties were investigated in skinned smooth muscle of the guinea pig Taenia coli. A "high" rigor state was obtained by removing MgATP at the plateau of an active contraction. Rigor force decayed slowly towards an apparent plateau of approximately 25-35% of maximal active force. MgPPi markedly increased the rate of force decay. The initial rate of the force decay depended on [MgPPi] and could be described by the Michaelis-Menten equation with a dissociation constant of 1.6 mM. The decay was irreversible amounting to approximately 50% of the rigor force. Stiffness decreased by 20%, suggesting that the major part of the cross-bridges were still attached. The results can be interpreted as "slippage" of PPi-cross-bridges to positions of lower strain. The initial rate of MgPPi-induced force decay decreased with decreasing ionic strength in the range 45-150 mM and was approximately 25% lower in thiophosphorylated fibers. MgADP inhibited the MgPPi-induced force decay with an apparent Ki of 2 microM. The apparent Km of MgATP for the maximal shortening velocity in thiophosphorylated fibers was 32 microM. This low Km of MgATP suggests that steps other than MgATP-induced detachment are responsible for the low shortening velocity in smooth muscle. No effects were observed of 4 mM MgPPi on the force-velocity relation, suggesting that cross-bridges with bound MgPPi do not constitute an internal load or that binding of MgPPi is weaker in negatively strained cross-bridges during shortening.     

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.     

P2Y1 receptor activation by photolysis of caged ATP enhances neuronal network activity in the developing olfactory bulb

It has recently been shown that adenosine-5'-triphosphate (ATP) is released together with glutamate from sensory axons in the olfactory bulb, where it stimulates calcium signaling in glial cells, while responses in identified neurons to ATP have not been recorded in the olfactory bulb yet. We used photolysis of caged ATP to elicit a rapid rise in ATP and measured whole-cell current responses in mitral cells, the output neurons of the olfactory bulb, in acute mouse brain slices. Wide-field photolysis of caged ATP evoked an increase in synaptic inputs in mitral cells, indicating an ATP-dependent increase in network activity. The increase in synaptic activity was accompanied by calcium transients in the dendritic tuft of the mitral cell, as measured by confocal calcium imaging. The stimulating effect of ATP on the network activity could be mimicked by photo release of caged adenosine 5'-diphosphate, and was inhibited by the P2Y(1) receptor antagonist MRS 2179. Local photolysis of caged ATP in the glomerulus innervated by the dendritic tuft of the recorded mitral cell elicited currents similar to those evoked by wide-field illumination. The results indicate that activation of P2Y(1) receptors in the glomerulus can stimulate network activity in the olfactory bulb.     

Allergic sensitization increases the amount of extracellular ATP hydrolyzed by guinea pig leukocytes

Purinergic Signalling - Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm...     

Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative

Na+-Dependent transmembrane transport of small neutral amino acids, such as glutamine and alanine, is mediated, among others, by the neutral amino acid transporters of the solute carrier 1 [SLC1, alanine serine cysteine transporter 1 (ASCT1), and ASCT2] and SLC38 families [sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4]. Many mechanistic aspects of amino acid transport by these systems are not well-understood. Here, we describe a new photolabile alanine derivative based on protection of alanine with the 4-methoxy-7-nitroindolinyl (MNI) caging group, which we use for pre-steady-state kinetic analysis of alanine transport by ASCT2, SNAT1, and SNAT2. MNI-alanine has favorable photochemical properties and is stable in aqueous solution. It is also inert with respect to the transport systems studied. Photolytic release of free alanine results in the generation of significant transient current components in HEK293 cells expressing the ASCT2, SNAT1, and SNAT2 proteins. In ASCT2, these currents show biphasic decay with time constants, tau, in the 1-30 ms time range. They are fully inhibited in the absence of extracellular Na+, demonstrating that Na+ binding to the transporter is necessary for induction of the alanine-mediated current. For SNAT1, these transient currents differ in their time course (tau = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (tau approximately 4-5 ms), indicating that they are associated with a fast, previously undetected, electrogenic partial reaction in the SNAT1 transport cycle. The implications of these results for the mechanisms of transmembrane transport of alanine are discussed. The new caged alanine derivative will provide a useful tool for future, more detailed studies of neutral amino acid transport.     

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects.

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.     

Relaxation of guinea pig trachealis during electrical field stimulation increases with age.

Our laboratory has previously shown that maturation of airway smooth muscle (ASM) contractility may play a role in the airway hyperresponsiveness displayed by juveniles of many species, including humans (Chitano P, Wang J, Cox CM, Stephens NL, and Murphy TM. J Appl Physiol 88: 1338-1345, 2000). ASM relaxation, which could also contribute to airway hyperresponsiveness, has neither been described nor quantified during maturation. Therefore, we studied ASM relaxation during and after electrical field stimulation (EFS) in tracheal strips from 1-wk-old, 3-wk-old, and 3-mo-old guinea pigs. Strips were stimulated (60 Hz, 18 V) at their optimal length for 15, 20, and 25 s, with and without the cyclooxygenase inhibitor indomethacin. To evaluate the role of the epithelium, deepithelialized strips from adult animals were also studied. New indexes were developed to quantify relaxation during EFS. We measured the time course of tension relaxation and its maximum rate (RTR) during the EFS, as well as the residual tension at the end of the EFS (TCT(end)). After EFS, we measured the maximum RTR and the time needed to reduce to half the TCT(end). Relaxation during the EFS significantly increased with age. Indomethacin reduced this age difference by increasing relaxation in strips from younger animals. By contrast, removal of the epithelium in adult strips decreased relaxation. Relaxation after EFS decreased with age and was not affected by indomethacin. In adult strips, it was further reduced by epithelium removal. Our results show that during EFS 1) airway smooth muscle relaxation increases with age, 2) cyclooxygenase metabolites oppose relaxation in younger animals, and 3) epithelium removal inhibits relaxation. We suggest that a reduced ASM relaxing ability during stimulation may be involved in juvenile airway hyperresponsiveness.