Differences in maximal activation properties of skinned short- and long-sarcomere muscle fibres from the claw of the freshwater crustaceanCherax destructor

Summary Single fibres of different sarcomere length at rest have been isolated from the claw muscle of the yabby (Cherax destructor), a decapod crustacean. Fibres of either long (SL > 6 m) or short (SL < 4 m) sarcomere length have been mechanically skinned and were maximally activated by Ca²⁺ and Sr²⁺ under various experimental conditions (ionic strength, in the presence of 2,3 butanedione monoxime (BDM)) to determine differences in their contractile properties. Isometric force was measured simultaneously with either myofibrillar MgATPase or fibre stiffness in both fibre types. The ultrastructure of individual long- and short-sarcomere fibres was also determined by electron microscopy. The long-sarcomere fibres developed greater tension (30.48±1.72 N cm^–2) when maximally activated by Ca²⁺ compared with the short-sarcomere fibres (18.60±0.80 N cm^–2). The difference in the maximum Ca²⁺-activated force can be explained by the difference in the amount of filament overlap between the two fibre types. The maximum Ca²⁺-activated myofibrillar MgATPase rate in the short-sarcomere fibres (1.60±0.27 mmol ATP l^–1 s^–1) was higher, but not significantly different from the ATPase rate in fibres with long-sarcomeres (1.09±0.14 mmol ATP l^–1 s^–1). As the concentration of myosin is estimated to be higher only by a factor of 1.22 in the short-sarcomere preparations there is no evidence to suggest that the myofibrillar MgATPase activity is different in the long- and short-sarcomere preparations. The maximum Ca²⁺-activated force (P ₀) of both short- and long-sarcomere fibres was quite insensitive to BDM compared with vertebrate muscle. Force decreased to 60.2±5.3% and 76.1±2.7% in the short- and long-sarcomere fibres respectively in the presence of 100 mmol l^–1 BDM. The difference in the force depression between the. long- and short-sarcomere fibres is statistically significant (p<0.05). Fibre stiffness during maximum Ca²⁺-activation expressed as percentage maximum force per nm per half sarcomere was higher by a factor of 3.5 in short-sarcomere fibres than in long-sarcomere fibres suggesting that the compliance of the filaments in the long-sarcomere fibres is considerably higher than in the short-sarcomere fibres. Sr²⁺ could not activate the contractile apparatus to the same level as that seen by Ca²⁺ in either fibre type: the maximum Sr²⁺-activated force was (20±3%) and (63±3%) of the maximum Ca²⁺-activated force response in short- and long-sarcomere fibres, respectively. The ratio between fibre stiffness in the maximum Sr²⁺-activating solution and the Ca²⁺-activating solution was very similar to the ratio between the maximum Sr²⁺-activated force and Ca²⁺-activated force in either type of fibres, suggesting that the number of attached crossbridges is lower in the fibres when maximally activated by Sr²⁺ than when maximally activated by Ca²⁺. The short-sarcomere fibres were also more sensitive to changes in ionic strength than long-sarcomere fibres. In conclusion these results indicate that while several important specific characteristics of the short- and long-sarcomere length fibres (ATPase, maximum Ca²⁺-activated force and fibre stiffness) can be explained solely on differences in the ultrastructure (length and density per cross-sectional area of myosin filaments) there are also differences in the properties of the proteins involved in the force production and regulation evidenced by the differential effect of Sr²⁺, BDM and ionic strength on contractile activation in the two fibre types.     

Effects of 2,3-butanedione monoxime on activation of contraction and crossbridge kinetics in intact and chemically skinned smooth muscle fibres from guinea pig taenia coli

Summary The effects of 2,3-butanedione monoxime (BDM) were studied in smooth muscle fibres from guinea pig taenia coli. In intact muscle, active force during contractions induced by high-K⁺ was inhibited by about 10% in 1 mM BDM and by approximately 70% in 10 mM BDM. Intracellular [Ca²⁺] during contraction, measured with the fura-2 technique, was reduced in the presence of BDM. The reduction in force and [Ca²⁺] in the presence of 1 and 10 mM BDM could be reproduced by reduction in extracellular Ca²⁺, suggesting that BDM influences the Ca²⁺ entry or release. In skinned muscle preparations, BDM decreased the Ca²⁺ sensitivity of active force. This change could be explained by a decreased level of myosin light chain phosphorylation. In fibres maximally activated by thiophosphorylation, the effect of BDM on force occurred at higher concentrations; 10 mM gave no reduction of force and 60 mM 15% reduction. The maximal shortening velocity (V _max) and force were unaffected by 30 mM BDM in thiophosphorylated muscle and decreased almost in parallel in Ca²⁺-activated contractions. The present results suggest that BDM inhibits myosin light chain phosphorylation, directly decreases force generation at the crossbridge level and inhibits the Ca²⁺ translocation in smooth muscle. The effect on force in skinned fibres is observed at higher BDM concentrations than those reported to be required for inhibition of force in striated muscle. The inhibition of force in intact smooth muscle could be explained by an influence on Ca²⁺ translocation.     

Potentiation of sarcoplasmic reticulum Ca2+ release by 2,3-butanedione monoxime in crustacean muscle

The effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on various aspects of excitation/contraction coupling in crustacean muscle was investigated. Despite having a depressant effect on vertebrate skeletal and cardiac muscle, BDM was a potentiator of contraction in crustacean muscle. At concentrations of 1–3 mM BDM caused an increase of potassium contractures in bundles of fibers isolated from crayfish muscle. At higher concentrations BDM caused oscillatory contractions by itself. In single voltage-clamped cut muscle fibers loaded with rhod-2, BDM (0.5–2 mM) potentiated the magnitude and duration of intracellular Ca²⁺ transients elicited by depolarization. At the same time BDM did not affect the rate of Ca²⁺ removal from the myoplasm under conditions where Ca²⁺ release was blocked by tetracaine. Nor did BDM increase Ca²⁺ entry; in fact it caused a decrease in the amplitude of the inward Ca²⁺ current (I _Ca). In microsomes isolated from lobster muscle, BDM also potentiated Ca²⁺ release induced by caffeine and at higher concentrations (above 3 mM) induced release by itself. At the same time it had little effect on Ca²⁺ uptake. These results indicate that BDM potentiates Ca²⁺ release in crustacean muscle possibly by dephosphorylation of the Ca²⁺-release channel.     

Effects of 2,3-butanedione monoxime on smooth-muscle contraction of guinea-pig portal vein

Effects of 2,3-butanedione-2-monoxime (BDM) on the contraction of intact and skinned smooth muscles from guinea-pig portal vein were examined. In intact preparations loaded with fura-2, 5–10 mM BDM markedly suppressed Ca²⁺ transients and force developments induced by 154 mM potassium and by phenylephrine (0.1 mM). On the other hand, in Ca²⁺-free depolarizing solution, BDM did not suppress phenylephrine (0.1 mM)-induced Ca²⁺ transient and force development. In skinned preparations obtained with Staphylococcus aureus -toxin treatment, BDM did not markedly affect active force development. The above results indicate that BDM suppresses contraction of the portal vein mainly by the inhibition of voltage-dependent cytosolic Ca²⁺ transients. An additional result suggests that BDM suppresses the force-enhancing effect of ₁-adrenergic agents on the contractile elements.     

Differential,direct effects of H+ on Ca2+-activated force of Skinned fibers from the soleus,cardiac and adductor magnus muscles of rabbits

The effect of acidosis on Ca²⁺-activated force generation was studied in rabbit soleus, left ventricular, and adductor magnus muscles. Fibers were skinned (sarcolemma peeled off or mechanico-chemically disrupted) to facilitate direct manipulation and standardization of their intracellular ionic milieus according to bathing solution composition. Skinned single skeletal and small bundles of cardiac fibers were mounted in a photodiode force transducer and activated by immersion in buffered-Ca²⁺ bathing solutions. The magnitude of steady state isometric force at each [Ca²⁺] was determined at pH 7.0 and 6.5 (paired data) at both 1 mM and 10 mM Mg²⁺ in order to detect artifacts of errors in calculated [Ca²⁺]. All bathing solutions contained: 7 mM total EGTA [ethyleneglycol-bis-(-amino-ethylether)-N,N tetra-acetic acid], 70 mM (Na⁺+K⁺), 2 mM MgATP^2– (Mg adenosine triphosphate), 15 mM CP^2– (creatine phosphate), 15 units/ml CPK (creatine phosphokinase), imidazole (adjusted ionic strength to 0.15 M), and propionate anion at 23±1° C. Maximum tensions were similar at both [Mg²⁺]s but less at pH 6.5 than at pH 7.0, with the following order of mean magnitude of acidotic depression adductor>cardiac>soleus. The proportionately greater acidotic depression of submaximum (relative to maximum) forces that occurred only at 1 mM Mg²⁺ (cardiac>adductor>soleus) implicates acidotic depression of Ca²⁺-activated force as a major cause of decreased cardiac contractility.Supported by National Institute of Health grants HL 17373 and RR00374. Preliminary report: Biophysical J. (abs)17, 201a (1977)Dr. Hermansen was a visiting scientist supported by Fogarty International Fellowship Grant TWO2230 from the National Institutes of Health and by the Perkins Fund of the American Physiological Society. Present Address: Institute of Work Physiology, Oslo, Norway     

2,3‐Butanedione monoxime increases speed of relaxation in single muscle fibres of frog

The effects of 2,3‐butanedione monoxime (BDM) on intracellular Ca²⁺ transient and cross‐bridge function were studied in frog single fibres from the anterior tibialis muscle of Rana temporaria (sarcomere length, 2.2 μm; temperature, 2–4 °C). The fluorescent dye fluo‐3 was used to monitor the intracellular free calcium concentration ([Ca²⁺]_i) during isometric contractions. BDM (1–5 mM ) reduced the amplitude of the Ca²⁺ transient during twitches, but this effect was too small to explain the marked inhibition of BDM on twitch force. [Ca²⁺]_i reached at the end of 1‐s tetanic stimulation was not significantly affected by BDM (1.0 and 1.8 mM ) while the maximum tetanic tension was substantially reduced. The rate of relaxation during isometric tetanus was increased by BDM whereas the rate of decay of the Ca²⁺ transient was reduced in the presence of BDM. The results strongly suggest that BDM, under the experimental conditions used, mainly affects the contractile machinery resulting in altered performance of the cross‐bridges. These effects of BDM were evaluated in terms of the cross‐bridge model of 17 which was fitted to the experimental force–velocity data in the presence and absence of BDM.     

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

The effects of orthovanadate (V_i), inorganic phosphate (P_i) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca²⁺ activation isometric force was depressed by V_i (0.03–10 mM), P_i (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1–0.3% of fibre length) were accelerated substantially by 1 mM V_i, 10 mM P_i or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca²⁺ removal after maximal Ca²⁺ activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V_i (1–10 mM), P_i (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V_i, P_i and BDM.     

Force-velocity relation in chemically skinned rat portal vein

Rat portal veins were chemically skinned using Triton X-100 and mounted for isometric and quick release experiments at 20°C. The skinned preparations were activated by Ca²⁺ (EGTA-buffered) in solutions containing 2 mM free-Mg²⁺ and 1 M calmodulin. Half maximal isometric force was obtained at pCa=6.2. Maximal force of the skinned preparations, at pCa=4.5, was 8.2±0.8 mN/mm² (n=6). Force-velocity relations were determined at varied Ca²⁺-concentrations. Maximal shortening velocity (V _max) was 0.10±0.01 lengths/s at pCa=4.5. At decreasing Ca²⁺-levelsV _max decreased (at pCa=6.25,V _max=0.05 l/s). At pCa =9 an increased level of free-Mg²⁺ (15mM) induces a slow and submaximal increase in tension. Force velocity relations of Mg²⁺-induced contractures were not different from those of Ca²⁺-contractures of similar magnitude (pCa=6.3). The results indicate that the degree of activation of the contractile system, as regulated by Ca²⁺ and Mg²⁺, influences the kinetic properties of the actomyosin interaction as well as the force development.     

The effects of 2,3-butanedione monoxime on initial heat,tension, and aequorin light output of ferret papillary muscles

At low concentrations (up to 5 mM) the compound 2,3-butanedione monoxime (BDM) was found to reduce twitch tension and initial heat production in isolated papillary muscles without significantly affecting the size of the intracellular Ca transient measured with aequorin luminescence. Higher concentrations of BDM caused further inhibition of twitch tension and heat production with a fall in the size of the Ca⁺ transient. The size of the aequorin transient was 50% of the control value at 15 mM BDM while twitch tension was negligible. These results suggest that BDM selectively inhibits Ca²⁺ activated force in cardiac muscle at low concentrations with additional effects on intracellular calcium at concentrations above 5 mM.     

Mechanical characteristics of chemically skinned guinea-pig taenia coli

Strips of intact and chemically skinned (Triton X-100) taenia coli were mounted for isometric and quick-release experiments at 23°C. Active force increased in repeated high-K⁺ induced contractures in the intact muscle. Stable maximal force was 313±24 mN/mm² (n=6). The skinned preparations activated by Ca²⁺, at 2 mM Mg²⁺, 3.2 mM MgATP and ionic strength 0.085 M, gave half maximal force atpCa=5.62±0.4 and a maximal force (63±8 mN/mm²) atpCa=4.5 (20–25 of the control K⁺-responses prior to skinning but about 60% of the first K⁺-response). Force-velocity relations were obtained from intact muscles and from the same muscles chemically skinned and activated at optimal Ca²⁺. Maximal shortening velocity (V _max) was unaltered in the skinned preparation compared to the intact muscle (0.138±0.011 vs 0.140±0.006 L/s) indicating similar kinetics of actomyosin interaction. In the intact muscle a decrease inV _max was found when the Ca²⁺ concentration was reduced. Calmodulin (1M) increased Ca²⁺ sensitivity (by about 0.6 log units) of the skinned preparation but at optimal Ca²⁺ caused no alteration in isometric force orV _max Apreliminary report of some of the results presented here was given at the Scandinavian Physiology Society Meeting in Århus, November 1981. Arner A, Hellstrand P (1982) Acta Physiol Scand (Abstract) 114: 38 A.     

The effects of caffeine and Ca2+ on rigor tension in triton-treated rat ventricular trabeculae

Ventricular trabeculae from rat heart were chemically skinned with Triton-X100, which disrupts all cellular membranes including the sarcoplasmic reticulum. Trabeculae developed a maintained rigor contracture when adenosine triphosphate was withdrawn from the bathing medium. In all preparations, the final level of rigor force developed in the presence of caffeine (10–40 mM) was greater than under control conditions. However, caffeine failed to increase rigor tension when applied after contracture had fully developed. The effect of caffeine on rigor was maximal at about 15 mM; concentrations greater or less than 15 mM were less effective. On average, caffeine decreased the time required to develop half-maximum rigor force. The caffeine-induced potentiation of rigor force occurred in the effective absence of Ca²⁺ (10^–9 M), in solutions strongly Ca²⁺-buffered with [ethylenebis(oxonitrilo)]tetraaceticacid (10–50 mM). In all preparations, rigor force was found to be independent of [Ca²⁺] over the range 10^–10 M to about 10^–7 M. These results suggest that caffeine affects rigor force by a direct effect on the myofilaments via a mechanism that is independent of Ca²⁺.     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.     

Low Ca2+-sensitive maxi-K+ channels in human cultured fibroblasts

The patch clamp technique was used to reveal single channel activity in the membrane of human cultured fibroblasts. The most frequently detected ion channel type was a Ca²⁺-dependent K⁺ channel with a conductance of 287±38 pS in symmetrical 130 mM KCl. The channel showed a peculiar low Ca²⁺-sensitivity compared to that of similar channels in other preparations. In fact micromolar values of internal Ca²⁺ were not effective in the channel activation, except at high depolarizing membrane potentials. The activity was highly increased only when the channel was exposed to relatively high internal Ca²⁺ concentrations (0.2–2.0 mM).     

Myofibrillar Ca2+ sensitivity of cardiomyopathic hamster hearts

We studied the Ca²⁺ responsiveness of skinned muscle fibre preparations from the right and left ventricles of normal (FIB) and genetically cardiomyopathic (Bio-To-2) Syrian hamsters. Thus, we compared the Ca²⁺/force relationships of preparations from myopathic hamsters to those of age-matched (11–16 months old) normal animals. The pCa (i.e. –log₁₀[Ca²⁺]) required for 50% force activation (Ca²⁺ sensitivity) was higher in the myopathic hamsters than in controls (pCa₅₀ values of 5.3±0.03 and 5.17±0.04, respectively); this difference might be due to an alteration in regulatory proteins. Indeed, after extraction (with vanadate) and replacement of troponin I with bovine cardiac troponin the pCa₅₀ values were similar (pCa 5.35) to those of bovine ventricular fibres. The Ca²⁺ sensitizer EMD 53998 (10 M) increased Ca²⁺ sensitivity in preparations from normal and cardiomyopathic hamsters equally, by 0.4 pCa units. Incubation of fibre bundles with the catalytic subunit of cyclicadenosine-monophosphate-dependent protein kinase decreased Ca²⁺ sensitivity, thereby normalizing the enhanced Ca²⁺ responsiveness of fibres from cardiomyopathic hamsters. It is not clear, however, whether the pathologically increased Ca²⁺ sensitivity of the hearts of aged myopathic hamsters reflects a maladaptation, or a compensatory mechanism of the failing heart.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca²⁺]_o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca²⁺ ([Ca²⁺]_i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential E_rev shifted from –2.3±0.8 mV (n=4) in a nominally Ca²⁺ free solution to +1.5±1.6 mV at 1.5 mM [Ca²⁺]_o (n=4) and to +21.9±4.4 mV (n=7) at 10 mM [Ca²⁺]_o. From our data we conclude that shear stress opens an ion channel that is 12.5±2.9 (n=7) times more permeable for calcium than for sodium or cesium.     

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes

 The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca²⁺]_i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca²⁺]_i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca²⁺]_i produced by caffeine. In voltage-clamped cells the rise of [Ca²⁺]_i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM. Received: 27 March 1998 / Received after revision: 12 May 1998 / Accepted: 13 May 1998