Kinetics of actin monomer exchange at the slow growing ends of actin filaments and their relation to the elongation of filaments shortened by gelsolin

Summary The kinetics of actin monomer exchange with the slow growing pointed ends of actin filaments have been determined by measuring rates of monomer addition to or loss from filaments with their fast-growing barbed ends blocked by the protein gelsolin. Direct measurements of filament length by electron microscopy confirmed that each gelsolin acts as a nucleus for an actin filament. The rate constants ascertained arek _–=0.03s^–1;k ₊=0.06 m ^–1 s^–1 at 23° C andk _–=0.11 s^–1;k ₊=0.09 m ^–1s^–1 at 37° C. They are approximately independent of pH from 7.0 to 8.0 at both temperatures. These rates are far slower than those reported on the basis of some electron microscopic studies of filaments assembled on to actin bundles. The rate constants also predict a higher critical monomer concentration for the pointed end at 37° C than at room temperature, consistent with direct measurements of this quantity. The relative slowness of the monomer exchange at the pointed end suggests that actin filaments with blocked barbed ends are relatively stable. The rate of redistribution of actin monomers from filaments stabilized at their barbed ends by the gelsolin-calcium complex to longer filaments was measured following removal of Ca²⁺, which decreases the capacity of gelsolin to nucleate filaments. The elongation occurs at a rate consistent with the measured rates of monomer exchange and is quantitatively described by Hill's model for filament elongation by random exchange of monomers from one end.     

Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle

Summary We have investigated the conditions for isolation of Ca²⁺-regulated thin filaments from sheep aorta. Inhibition of proteolysis by 2 µg ml^–1 leupeptin and chymostatin and of oxidation with 5mm dithiothreitol were essential. Washed homogenates were extracted in 10mm ATP of low ionic strength at pH 6.1 to minimize coextraction of myosin with thin filaments. Thin filaments were separated from myosin by high speed sedimentation; 20% glycol was added to prevent loss of regulatory factors and tropomyosin. The resulting thin filaments (yield 2.5 mg protein g^–1 artery wet weight) were made up of actin, tropomyosin and a 120 000M _r protein (molar ratio 1:1/5:1/29) and were up to 4 µm long. They activated skeletal muscle myosin at least 50 times in presence of Ca²⁺. Up to 80% inhibition was observed in the absence of Ca²⁺. We also prepared pure arterial F-actin, which activated skeletal myosin more than the thin filaments, but was similar to skeletal F-actin. We conclude that Ca²⁺ regulation is negative, involves cooperative interactions between actin, myosin and tropomyosin and suggest that it is mediated by the 120 000M _r protein.     

The effects of a 45 000 molecular weight protein from unfertilized sea urchin eggs and its 1:1 actin complex on actin filaments

Summary A 45 kDa actin-binding protein (SU45) has been isolated previously from egg extracts of the Hawaiian sea urchinTripneustes gratilla by DEAE Sephacel, Sephadex G-75 and hydroxyapatite chromatography. Using pyrene-labelled rabbit skeletal muscle actin, we have found that when SU45 is added to actin in the presence of calcium and the salt concentration is increased, the initial rate of actin assembly is accelerated. Moreover, the final polymer concentration is reduced indicating that SU45 caps the preferred end of actin filaments shifting the critical concentration (C_c) to that of the nonpreferred end. Determination of the C_c as a function of the concentration of SU45 gave an apparent K_D of 1nm. Dilution of F-actin to below its C_c, into buffers containing SU45 and Ca²⁺ resulted in a sharp increase in the rate of depolymerization; reducing the Ca²⁺ concentration attenuated this effect. Incubation of SU45 with rabbit skeletal muscle G-actin yielded a 1:1 complex which held⁴⁵Ca²⁺ tightly with a dissociation half-time of 10.8 days. By kinetic analyses of assembly in the presence of the SU45-actin complex and dilution-induced disassembly of filaments precapped with complex, we have estimated both the association rate constant (4.0×10⁴ m^–1 s^–1) and the dissociation rate constant (0.05 s^–1) for the nonpreferred ends of actin filaments. Finally, dilution of F-actin to below its C_c, into complex in either Ca²⁺ or EGTA resulted in a much slower depolymerization consistent with a rapid capping of the preferred end by the SU45-actin complex.     

Tension generation and relaxation in single myofibrils from human atrial and ventricular myocardium

Fast solution switching techniques in single myofibrils offer the opportunity to dissect and directly examine the sarcomeric mechanisms responsible for force generation and relaxation. The feasibility of this approach is tested here in human cardiac myofibrils isolated from small samples of atrial and ventricular tissue. At sarcomere lengths between 2.0 and 2.3 μm, resting tensions were significantly higher in ventricular than in atrial myofibrils. The rate constant of active tension generation after maximal Ca²⁺ activation (k _ACT) was markedly faster in atrial than in ventricular myofibrils. In both myofibril types k _ACT was the same as the rate of tension redevelopment after mechanical perturbations and decreased significantly by decreasing [Ca²⁺] in the activating solution. Upon sudden Ca²⁺ removal, active tension fully relaxed. Relaxation kinetics were (1) much faster in atrial than in ventricular myofibrils, (2) unaffected by bepridil, a drug that increases the affinity of troponin for Ca²⁺, and (3) strongly accelerated by small increases in inorganic phosphate concentration. The results indicate that myofibril tension activation and relaxation rates reflect apparent cross-bridge kinetics and their Ca²⁺ regulation rather than the rates at which thin filaments are switched on or off by Ca²⁺ binding or removal. Myofibrils from human hearts retain intact mechanisms for contraction regulation and tension generation and represent a viable experimental model to investigate function and dysfunction of human cardiac sarcomeres.     

Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils

The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (k _act), calcium sensitivity (pCa₅₀), and cooperativity (n _H). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Δ23Tm, a deletion mutant lacking the second and third of Tm’s seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower k _act rates and reduced n _H. Δ23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Δ23Tm also were investigated using P _i and ADP jumps. P _i and ADP kinetics were indistinguishable in Δ23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis

Summary Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determinein situ the concentrations of Ca²⁺ and Mg²⁺ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2×10^–11 m Ca²⁺ and 2.7×10^–9 m Mg²⁺, the inexchangeably bound Mg²⁺ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca²⁺ bound to the I-band was not significantly above zero. The bound Mg²⁺ in the I-band was not exchangeable with Ca²⁺ even when the skinned fibres were exposed to 10mm Ca²⁺ solution. These results clearly indicate that Mg²⁺, rather than Ca²⁺, is the divalent cation bound to F-actin in the thin filamentsin situ. In the presence of 1mm Mg²⁺, the exchangeable Ca²⁺ bound to the I-band was increased as a function of the free Ca²⁺, while that in the A-band was not significantly changed with [Ca²⁺] up to 2 × 10^–5 m, and increased to approximately 0.8 mol Ca²⁺ per mol myosin at 10^–4 m Ca²⁺. At a saturating free Ca²⁺ in Tris-Cl solution, the bound Ca²⁺ content (2–3 mol Ca²⁺ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na⁺ enhanced Ca²⁺ binding to the level equivalent to 3–4 mol Ca²⁺ per mol troponin. The depressant effect of Tris on Ca²⁺ binding was greater in the absence of Mg²⁺. High concentrations of Tris also reduced the maximum tension induced by 10^–4 m Ca²⁺ buffered with 10mm EGTA. At 1.3×10^–7 m Ca²⁺, thought to be close to the cytoplasmic free Ca²⁺ in resting muscle, the I-band bound a significant amount of Ca²⁺: equivalent to about 1 mol Ca²⁺ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca²⁺ bound by the A-band at a free Ca²⁺ of 10^–4 m.     

Actin cytoskeleton role in the structural response of epithelial (MDCK) cells to low extracellular Ca2+

Kinetic and stereometric assessment of the mechanical responses of epithelial cells to variations in the concentration of extracellular Ca²⁺ was carried out in vivo at the single cell level. Continuous monitoring of individual MDCK cells in subconfluent cultures attested to an intense, immediately relaxable, and cytochalasin D-sensitive contraction, equivalent to that seen in confluent monolayers following depletion of external Ca²⁺ (<0.1 mM). Increasingly greater and less readily reversible contractions were performed upon repeated stimulation with short-term cycles of alternating normal (30 min) and low Ca²⁺ (30 min) media. Constriction of a narrow horizontal girdle corresponding in position to the major ring-like bundle of actin filaments eventually develops into a deep lateral furrow in intensely contracted cells. Substantial membrane infolding in the contracted state is indicated also by stereometric estimates of apparent bounding surface area. Irrespective of the contracted or relaxed cell condition, rhodamine–phalloidin labeling showed a marginal position of the ring-like bundle of microfilaments and other components of the actin cytoskeleton. These results suggest, contrary to prevalent views, that the actin–myosin system stays associated to the cortex and retains contractile capability in epithelial cells deprived of external Ca²⁺. Hence, the mechanical responses to variations of Ca²⁺ may be an overstrained expression of a physiological mechanism.     

Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Summary The direct binding of Ca²⁺ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnC _DANZ ) was measured (25° C) at three Mg²⁺ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca²⁺ to high- and low-affinity sites of troponin. The binding of Ca²⁺ to the high-affinity sites had a greater sensitivity to Mg²⁺ (K _Mg=1×10⁴ m ^–1) than the low-affinity sites (K _Mg=1.2×10³ m ^–1). The fluorescence change of thin filaments reconstituted with TnC _DANZ was titrated with Ca²⁺ in the same solutions used for binding assays. The Ca²⁺-dependent fluorescence change had nearly the same sensitivity to Mg²⁺ (K _Mg=9.4×10² m ^–1) as did Ca²⁺ binding to the low-affinity sites. The Ca²⁺ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca²⁺ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca²⁺ binding to the low-affinity sites of isolated TnC _DANZ was measured (4° C). From these results the binding of Ca²⁺ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca²⁺ affinity.     

Swelling of rat mesangial cells induces a Ca2+-dependent Cl− conductance

Membrane voltage (V _m) and ion currents of rat mesangial cells in primary culture were measured with the patch-clamp technique in the fast whole-cell configuration.V _m was –44 ± 1 mV (_n = 138). A reduction of the osmolality from 290 to 190 mosmol/kg depolarizedV _m from –44 ± 1 to –29 ± 1 mV (_n = 118) and increased the inward and outward conductances (Gm) from 14±2 to 39 ± 4 nS and 13±2 to 37 ± 4 nS (_n = 84), respectively. During the hypotonicity-induced depolarization the cell capacitance increased significantly from 33 ± 3 to 42 ± 4 pF (_n = 40). The effect of hypotonic cell swelling onV _m was increased in a bath with a reduced extracellular Cl^– of 32 mmol/l (by 71 ± 4%,_n = 23), indicating that a Cl^– conductance was activated. The permselectivity of this conductance was I^– Br^– > Cl^–. TheV _m response was not affected in the presence of a reduced extracellular Na⁺ of 5 mmol/l (n = 13) and was inhibited in a solution with reduced extracellular Ca²⁺ concentration (by 63 ± 9%,n = 14). In microfluorescence measurements with the Ca²⁺-sensitive dye fura-2 hypotonic cell swelling induced a sustained increase of the intracellular Ca²⁺ activity, [Ca²⁺]_i (_n = 19). The increase of [Ca²⁺]_i was completely inhibited when the extracellular solution was free of Ca²⁺. TheV _m response to hypotonic cell swelling was not attenuated in the presence of the L-type Ca²⁺ channel blockers nicardipine (n = 5), nifedipine (n = 5) and verapamil (n = 5) (all at 1 mol/l). The data indicate that in rat mesangial cells, osmotic swelling induces a Ca²⁺ influx from extracellular space. This Ca²⁺ influx activates a Cl^– conductance resulting in a depolarization ofV _m. The enhanced Cl^– conductance may lead to KCl extrusion and hence regulatory volume decrease.     

Kinetics of ATP release and Pi binding during the ATPase cycle ofLethocerus flight muscle fibres,using phosphate-water oxygen exchange

Summary Rate constants have been obtained using oxygen isotope exchange techniques for steps controlling ATP release and P_i binding in the ATPase cycle of insect flight muscle fibres from the giant waterbugLethecerus. The new exchange data for P_i binding and ATP release are compatible with a model developed previously in which only the rate constants controlling P_i and ATP release change during fibre activation.Phosphate-water oxygen exchange occurs into ATP remaining after partial hydrolysis by chemically skinned fibres in (¹⁸O) water. For fully activated fibres, the results are compatible with a single set of rate constants controlling this exchange and give a rate constant for ATP release of 1 s^–1 (21° C, pH 7.0I=120mm). Oxygen exchange also occurs between (¹⁸O₄)P_i in the medium and water during ATP hydrolysis. There is a strong correlation between the measured rate constant of exchange and the value ofk _cat for the ATPase activity at different levels of activation. For fibres fully activated by oscillation or strain, the rate constant for P_i binding to an actomyosin. ADP state is >960m ^–1 s^–1.     

The effect of MgATP on forming and breaking actin-myosin linkages in contracted skinned insect flight muscle fibres

Summary At neutral pH, fully Ca²⁺ -activated glycerinated dorsal longitudinal fibre bundles fromLethocerus indicus contract under isometric conditions and respond to release by deactivation, i.e. quick release causes a delayed tension fall. At slightly alkaline pH, the release-induced deactivation becomes a transient phenomenon, i.e. a delayed tension fall is followed by a slow tension recovery. This enabled us to study the effect of MgATP concentration on the phases of deactivation and slow recovery. Reduction of the MgATP concentration slows down the tension response to a quick length change and increases the time constants of the delayed deactivation phase and of the slow recovery phase. The rate constants depend on the ATP concentration according to the Michaelis-Menten law yielding apparent dissociation constants (K _m) of 2mm and 0.09mm and maximal rate constants of 700 s^–1 and 20 s^–1 for the deactivation phase (crossbridge detachment) and slow recovery phase (crossbridge reattachment) respectively. The rate of MgATP hydrolysis is also hyperbolically related to the MgATP concentration (K _m = 0.14mm, maximal MgATP turnover rate 1.2 s^–1).It is concluded that the effect of MgATP on the deactivation phase, in which crossbridges dissociate strain dependent from the actin, is controlled by at least two mechanisms: (1) fast equilibrium transitions within attached crossbridge states which augment MgATP dissociation from crossbridges with discharged elastic elements; and (2) a crossbridge strain-dependent isomerization of the ternary actin-myosin-MgATP complex which determines crossbridge detachment from the actin.     

Identification of functioning regulatory sites and a new myosin binding site in the C-terminal 288 amino acids of caldesmon expressed from a human clone

Summary A partial clone of caldesmon, coding for the C-terminal 288 amino acids, was isolated from a human fetal liver cDNA library and sequenced. Expression of the clone in Escherichia coli produced a peptide called H1 (M_r 32 549), which inhibited tropomyosin-enhanced actomyosin Mg²⁺-ATPase activity by 90% with half maximal inhibition at 0.03–0.04 mol H1 per mol actin. The inhibition could be reversed by Ca²⁺-calmodulin. H1 bound actin, Ca²⁺-calmodulin and tropomyosin and smooth muscle myosin with high affinities. This latter finding shows the presence of a second myosin-binding site in caldesmon. This was confirmed in thrombic digests of native sheep aorta and chicken gizzard caldesmon.Abbreviations DTT dithiothreitol - H1 expressed C-terminal 288 amino acids of human caldesmon - PMSF phenylmethanesulfonyl fluoride - TA 2,5-dichloro-triazinyl-(N,N-diethylamino-)benzene - TLCK 1 chloro-3-tosylamido-7-amino-L-2-heptanone     

Alpha-tropomyosin mutations in inherited cardiomyopathies

The inherited cardiac diseases hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) can both be caused by missense mutations in the TPM1 gene which encodes the thin filament regulatory protein α-tropomyosin. Different mutations are responsible for either HCM or DCM, suggesting that distinct changes in tropomyosin structure and function can lead to the different diseases. Various biophysical and physiological approaches have been used to investigate the structure–function effects of the mutations, and animal models developed. The reported effects of the mutations include changes to the secondary structure of tropomyosin, its binding to actin and its position on the thin filament, and alterations to actin–myosin interactions and myofilament Ca²⁺ sensitivity. The latter changes have been found to be particularly consistent, with HCM mutations increasing Ca²⁺ sensitivity and DCM mutations in general decreasing this parameter and uncoupling the effect of troponin phosphorylation upon Ca²⁺ responsiveness. As well as impacting on contractility, these changes are likely to alter intracellular Ca²⁺ handling and signaling, and a combination of these alterations may provide the trigger for disease remodeling.     

Effects of acidic stimuli on intracellular calcium in isolated type I cells of the neonatal rat carotid body

We have investigated the effects of acidic stimuli upon [Ca²⁺]_i in isolated carotid body type I cells from the neonatal rat using indo-1 (AM-loaded). Under normocapnic, non-hypoxic conditions (23 mM HCO₃ ^–, 5% CO₂ in air, pH_o=7.4), the mean [Ca²⁺]_i for single cells was 102±5.0 nM (SEM, n=55) with 58% of cells showing sporadic [Ca²⁺]_i fluctuations. A hypercapnic acidosis (increase in CO₂ to 10%–20% at constant HCO₃ ^–, pH_o 7.15–6.85), an isohydric hypercapnia (increase in CO₂ to 10% at constant pH_o=7.4) and an isocapnic acidosis (pH_o=7.0, constant CO₂) all increased [Ca²⁺]_i in single cells and cell clusters. The averaged [Ca²⁺]_i response to both hypercapnic acidosis and isohydric hypercapnia displayed a rapid rise followed by a secondary decline. The averaged [Ca²⁺]_i response to isocapnic acidosis displayed a slower rise and little secondary decline. The rise of [Ca²⁺]_i in response to all the above stimuli can be attributed to no single factor other than to a fall of pH_i. The hypercapnia-induced rise of [Ca²⁺]_i was almost completely abolished in Ca²⁺-free solution, suggesting a role for Ca²⁺ influx in triggering and/or sustaining the [Ca²⁺]_i response. These results are consistent with a role for type I cell [Ca²⁺]_i in mediating pH/PCO₂ chemoreception.     

Structural and functional reconstitution of thin filaments in skeletal muscle

Summary Thin filaments were reconstituted by incorporating exogenous actin, tropomyosin and troponin into glycerinated skeletal muscle fibres or myofibrils. Firstly, thin filaments except short fragments at the Z line were selectively removed by treatment with plasma gelsolin, an actin severing protein. As a result, the fibres (or fibrils) lost the ability to generate active tension. Next, actin filaments were reconstituted by adding purified G-actin which polymerizes onto the actin fragments which remained at the Z line. Rhodamine phalloidin staining of myofibrils showed that exogenous actin was incorporated into the position where the intrinsic thin filaments located. Thin section electron micrographs of fibres showed that reconstituted actin filaments ran from the Z line to the inside of the A band, with some reaching the H zone. The number density of reconstituted actin filaments in the A band was about 20% of that found in intact fibres. The actin filament-reconstituted fibres (or fibrils) generated active tension in a Ca²⁺-insensitive manner and the tension was reversibly suppressed by 2,3-butanedione 2-monoxime. The recovered active tension was about 20% of tension developed by intact fibres. These results indicate that reconstituted actin filaments bear active tension similar to that borne by intact thin filaments. Thin filament-reconstituted fibres, which were prepared by adding purified tropomyosin-troponin complexes into actin filament-reconstituted fibres, showed Ca²⁺-sensitive tension generation. The maximum tension generated was not affected by the presence of tropomyosin and troponin. SDS-PAGE analysis showed that more than 25% of actin and 20% of tropomyosin and troponin was incorporated into the reconstituted fibres. These results indicate that the structure and function of thin filaments are substantially reconstituted by self-assembly of actin, tropomyosin and troponin. The reconstituted fibres and fibrils will be useful for studying the molecular mechanism of muscle contraction and its regulation.     

A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate

 The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (P_i) and H⁺ ([MgADP], [Pi] and [H⁺], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k₁ [5.6±0.1 s^–1 for barnacle, n=30; 26.3±0.7 s^–1 for frog, n=14 (mean±SEM)] and k₂ [0.6±0.1 s^–1 in barnacle, n=30; 10.4±1.0 s^–1 in frog, n=14 (mean±SEM)], at 10°C. Decreasing the pH by 0.5 pH units did not significantly affect k₁ for barnacle relaxation [5.6±0.1 s^–1 (mean±SEM), n=15] compared to the decrease in k₁ of 40% seen in frog. Use of the Ca²⁺-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa₅₀ value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in k_off from the labelled protein [322±32 s^–1 at pH 7.0 and 381±24 s^–1 (mean±SEM) at pH 6.6]. Increasing [MgADP] by 20 μM (50 μM added ADP) from control values of 50 μM in frog decreased k₁ to 12.3±0.4 s^–1 (mean±SEM, n=8), and at 400 μM MgADP, k₁=9.6±0.1 s^–1 (mean±SEM, n=12). In barnacle, 500 μM MgADP had a much smaller effect on k₁ (4.0±0.9 s^–1, mean±SEM, n=8). Increasing the free [P_i] from the contaminant level of 0.36 mM to 1.9 mM slowed k₁ by ≈15% in barnacle [4.8±0.8 s^–1, mean±SEM, n=7], compared to a ≈30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation. Received: 18 May 1998 / Received after revision and accepted: / 1 September 1998     

Functional differences in type-I fibres from two slow skeletal muscles of rabbit

The present study addressed the question of whether the slow fibres of mammalian skeletal muscle, containing the myosin heavy chain MHCI (type-I fibres), are a functionally homogeneous population. We compared various properties of Ca²⁺-activated, skinned, type-I fibres from the soleus and semitendinosus muscles of a rabbit. Soleus type-I fibres showed significantly faster kinetics of stretch activation, measured as the time-to-peak of the stretch-induced, delayed force increase, t₃, than semitendinosus fibres (1239±438 ms, n=136, vs. 1600±409 ms, n=208 respectively) (means±SD, 22 °C). Similarly, the speed of unloaded shortening at 15 °C was faster in soleus than in semitendinosus fibres [0.79±0.16 fibre lengths (FL) s^–1, n=44, vs. 0.65±0.15 FL s^–1, n=35 respectively]. The kinetics of stretch activation were more temperature sensitive in semitendinosus than in soleus fibres. Finally, the generation of steady-state isometric force was more sensitive to Ca²⁺ in semitendinosus than in soleus fibres: [pCa₅₀ (–log [Ca²⁺] for half-maximal activation) at 22 °C: 6.29±0.15, n=28, vs. 6.19±0.10, n=18 respectively]. These results suggest strongly that there is no functional homogeneity within type-I fibres of different muscles. The observed differences might reflect the existence of more than one functionally different slow myosin heavy chain isoforms or other modifications of contractile proteins.     

Rate of active tension development from rigor in skinned atrial and ventricular cardiac fibres from swine following photolytic release of ATP from caged ATP

We investigated the rate of tension development (k_td) after photolytical release of ATP from, P³-l-(2-nitrophenyl)-ethyladenosine-5′-triphosphate (‘caged ATP’) of atrial and ventricular fibre bundles from pig. Contraction was initiated from high-tension (HT) and low-tension (LT) rigor at maximal Ca²⁺ activation (pCa 4.5). The k_td of atrial fibre bundles was 6.8 s^_1 from LT and 6.9 s^_1 from HT rigor. Rate of tension development of ventricular fibre bundles was significantly lower (P < 0.001) being 1.06 s“¹ and 0.94 s”¹ from LT and HT rigor, respectively. The k_td of skinned ventricular fibre bundles incubated in a high [K⁺], low [Ca²⁺] (cardioplegic) solution prior to the skinning procedure decreased significantly (P < 0.05) to 0.73 s^-1 and 0.63 s_¹ from LT and HT rigor, respectively, whereas that of skinned atrial fibre bundles remained at 7.1 s^-1 and 6.9 s^-1 from LT and HT rigor, respectively. Phosphorylation levels of the myosin light chain 2 isoform in the atrial fibre bundles (ALC-2) was 15.6±2.7%. The corresponding values for the two ventricular isoforms, VLC-2 and VLC-2*, were 31.2 ± 0.4% and 25.1 ±2.1%, respectively. Phosphorylation levels of fibre bundles incubated in cardioplegic solution prior to skinning were 11.6%, 18.9%, and 15.4% of the ALC-2, VLC-2 and VLC-2*, respectively. The results show that the rate of tension development is more than seven-fold higher in the atrial compared with ventricular fibre bundles. These results correlate with the differences in ATPase activity of the contractile proteins in solution and, most likely, reflect differences in the myosin isoform composition. In ventricular fibre bundles the increased levels of light chain phosphorylation were associated with increased rate of contraction.     

Recognition of the ‘calcium paradox’ and the effects of verapamil and gallopamil in human adenoidal mast cells

Human mast cells from adenoids show when resuspended in medium containing 10^–3 M CaCl₂ after their temporary exposure to Ca²⁺-free saline for about 20 min an irreversible reduction of responsiveness to a variety of stimuli: The histamine release induced by concanavalin A or ionophore A 23187 is only 30–50% of the one obtained in cells which were kept in 10^–3 M Ca²⁺ throughout the experiment. This phenomenon called calcium paradox can be almost entirely avoided if the cells are temporarily exposed to 10^–4 M Ca²⁺ instead of Ca²⁺-free saline. Number yields, average histamine contents of mast cells and the rate of the spontaneous histamine release are not affected by the transitory lack of Ca²⁺, nor is the histamine release enhancing effect of adenosine.At 10^–3 M Ca²⁺ concentration the calcium antagonists verapamil or gallopamil cause a significant inhibition of the Con A-induced histamine release only at concentrations much higher (10^–4 M) than those effective in smooth muscle preparations. The actions of both calcium antagonists were not affected by the presence of added extracellular adenosine.To whom requests for reprints should be addressed.