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.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

 The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca²⁺], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22° C. In the maximally thiophosphorylated muscle neither OA (10 μM) nor Ca²⁺ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca²⁺] in the presence of 0.5 μM calmodulin, both force and the maximal shortening velocity (V _max) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, V _max was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca²⁺] and at varied [Ca²⁺]. The relation between force and V _max when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca²⁺]. The results show that Ca²⁺ and OA do not influence force or V _max in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining V _max. The finding that the relationship between force and V _max was similar when activation was altered with OA and Ca²⁺ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres. Received: 1 December 1995 / Received after revision: 20 June 1996 / Accepted 12 July 1996     

Mechanics and Ca2+-sensitivity of human detrusor muscle bundles studied in vitro

Mechanical properties of isolated smooth muscle strips from human urinary bladder were investigated in vitro. Bladder tissue was obtained from tumour-free wall regions of bladders from male patients undergoing cystectomy for bladder carcinoma. In intact muscle strips, activated with high-K⁺ solution, half-maximal force occurred at about 0.9 mm extracellular [Ca²⁺]. The length-active force relation was determined and the muscle strips were fixed for light and electron microscopy at optimal length for active force (I_o). The maximal active force per unit smooth muscle cross-sectional area was 208±49 mN/mm², n= 6. Chemically skinned preparations were obtained by treatment with triton X-100. These preparations had a steep [Ca²⁺]-force relation in the micromolar range which was influenced by calmodulin. The skinned preparations could be maximally activated by irreversible thiophosphorylation of the regulatory light chains. The force-velocity relation was determined in the maximally activated skinned muscle at 22 °C at 0.5 1_o. When the muscle was shortened by 10%, force was reduced by 35% whereas the maximal shortening velocity was little affected.     

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.     

Structure and mechanics of growing arterial microvessels from hypertrophied urinary bladder in the rat

Rat bladder hypertrophy, induced by a partial ligation of the urethra, was used to study the accompanying changes of microvascular smooth muscle mechanics, pharmacology and morphology. A segment of a microarterial vessel to the bladder was taken from a defined anatomical location and studied in a wire myograph in vitro at the length for maximal isometric force development (L _max). After 10 days of ligation, bladder hypertrophy resulted in a microvascular growth response compared to non-operated controls which was characterized by (i) an increase of the calculated diameter at L _max from 134±5 m to 222±19 m; (ii) an increase of the media thickness from 22.4±1.9 m to 32.2±3.0 m; (iii) an increase of the active tension from 1.42±0.28 mN/mm to 3.06±0.33 mN/mm; (iv) no change of the wall/lumen ratio (from 0.83±0.10 to 0.79±0.15). Normalized length/force relations (active, passive and total) did not differ significantly between microarteries from control and hypertrophic bladders. Microvascular smooth muscle growth was also associated with a decreased sensitivity to K⁺-induced depolarization and an increased sensitivity to ₁-adrenergic stimulation. No differences were noted regarding the Ca²⁺ sensitivity of force during K⁺-induced depolarization. The results suggest that microvascular growth (1) is immediately and positively influenced by the organ growth; (2) results in a functional resetting of the microvascular segments towards larger diameters without gross morphological or mechanical alterations; and (3) is accompanied by pharmacological alterations of the smooth muscle reactivity.     

The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit

Summary The relationship between maximum shortening velocity (V _max) and free calcium concentration has been studied in skinned single fibres from rabbit psoas and soleus muscles. At both 10 and 15° C,V _max measured in the psoas fibres was found to decrease by 40% when the pCa (-log[Ca²⁺]) was increased from 5.49 (maximally activating) to 6.21. Further decreases inV _max were observed when the pCa was increased to 6.32.V _max measured in soleus fibres at 15° C also decreased when the Ca²⁺ concentration was lowered, though the magnitude of this effect was slightly less than in the psoas fibres. Thus, a distinct effect of Ca²⁺ uponV _max has been shown to occur in mammalian skeletal muscle. The occurrence of this effect in both fast and slow muscle types may indicate that the underlying mechanism in the two cases is similar.     

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.     

Regulation of force in skinned,single cells of ferret aortic smooth muscle

An isolation technique was developed for single cells from the ferret aorta, which resulted in the isolation of long (87±27 m; x±SD, n=62), relaxed, pharmacologically active smooth muscle cells. These cells were attached to microtools, one of which was connected to a force transducer. Force in maximally phenylephrine-stimulated contractions of the intact cells averaged 2.3 ±1.4 N (n=17). After cell skinning with saponin, the threshold for force development was 0.05 M [Ca²⁺], and force reached a maximum of 4.4±1.6 N (n = 36) at 0.5 M [Ca²⁺]. Plots of relative steady-state force vs pCa (–log₁₀[Ca²⁺]) were fit to the Hill equation, which yielded a pCa at half-maximal force of 6.87 ± 0.30 and a Hill coefficient of 2.3±1.4 (n = 29). When 2.5 M calmodulin was added to the solutions, the calcium sensitivity of force was significantly increased (P<0.05) without changing the maximal force (P>0.05). In a solution of pCa 7, the skinned cells developed 2.5±0.5 N (n = 5) of force when stimulated with a phorbol ester. The addition of a specific inhibitor (17 kDa) of protein kinase C to the calcium buffers depressed (P<0.05) the maximally Ca²⁺ -activated force without a change in the calcium sensitivity of force (P>0.05). These data strongly suggest that in vascular smooth muscle, protein kinase C may be involved in a physiological, regulatory system for force.     

The effect of temperature on contractile activation of intact and chemically skinned ‘catch’ muscle fibre bundles of Mytilus edulis

Summary The effect of temperature (5–35° C) on maximum force production was examined in intact and chemically skinned muscle fibre bundles (10–25 fibres) from the anterior byssus retractor muscle of Mytilus edulis. In intact fibre bundles, 10 m acetylcholine induced a tonic contraction which had a magnitude of 65.4±4.0 N cm^-2 (n=30) at 23° C. Activation by caffeine (20 mm) produced a force response which was 157.1±7.9% (n=16) of the acetylcholine response at 23° C and acetylcholine and caffeine together produced force which was not significantly different from the response to caffeine alone. At 5° C the acetylcholine and caffeine responses were decreased by 9.6±3.4% (n=6) and 14.6±2.8% (n=8) compared with the respective responses at 23° C. However, there was no significant reduction of the response induced by the combined action of acetylcholine and caffeine when the temperature was decreased from 23° C to 5° C. The 20–80% of peak force activation time increased by about one order of magnitude for all acetylcholine, caffeine and combined acetylcholine-caffeine-induced responses when the temperature was decreased from 23–5° C. Repeated exposure of the intact preparation to caffeine caused a marked decrease in the caffeine-induced response (complete abolition of force after the third exposure to caffeine), but the response to caffeine could be fully restored following one acetylcholine-induced activation. The maximum Ca²⁺-activated force after skinning the preparation with saponin was not significantly different from the caffeine or combined acetylcholine-caffeine-induced responses before skinning. In the saponin skinned fibre preparation a drop in temperature from 23° C to 15° C or 5° C decreased the maximum Ca²⁺-activated force by 13.2±1.4% (n=8) and 41.4±3.1% (n=5) respectively. The activation time between 20–80% of the peak Ca²⁺-activated force increased at 15° C and 5° C by a factor of 1.5±0.1 (n=5) and 6.8±1.1 (n=5) respectively when compared to corresponding values at 23° C. The relaxation half-time decreased by a factor of 1.7±0.2 (n=5) and 3.0±0.2 (n=5) at 15° C and 5° C respectively compared with that at 23° C. It was possible to distinguish between the temperature effects on the contractile apparatus per se and the Ca²⁺ regulatory system with the results indicating that the contractile apparatus was more sensitive to a change in temperature than the Ca²⁺-regulatory system. Increasing the temperature to 35° C irreversibly affected the ability to develop and maintain force in both intact and skinned muscle preparations. These results indicate that: (1) acetylcholine does not fully activate the intact catch muscle at 23° C; (2) acetylcholine is able to replenish the internal stores after depletion by caffeine; (3) compensatory mechanisms which oppose the inhibitory effect of lower temperatures on the contractile apparatus and the Ca²⁺-regulatory system must be operating in the intact fibre preparations.     

Calcium-activated and stretch-induced force responses in two biochemically defined muscle fibre types of the Norway lobster

Summary Mechanical properties of thin (<80 m) myofibrillar bundles from single rehydrated freeze-dried fibres of the superficial abdominal flexor muscle of the lobster Nephrops norvegicus have been measured, and subsequently the protein content of these fibres has been analysed by SDS-PAGE. Two slow fibre phenotypes can be distinguished on the basis of their myofibrillar assemblages and sarcomere length (type S₁: 6.0–7.5 m, type S₂: 8.0–10.9 m). Differences (means ± sd, average of seven fibres of each type) were observed in the kinetics for Ca²⁺ activation (half time of force development (ms); S₁: 416±174; S₂: 762±199 plus a delay of 280±130) and relaxation (half time of force decay (ms); S₁: 162±75, S₂: 257±53), for Ca²⁺ sensitivity of force generation (-log [Ca²⁺] for half maximal activation; S₁: 5.40±0.12; S₂: 5.55±0.08), and of the kinetics of stretch activation (delay of the peak of stretch-induced force increase (ms); S₁: 91±30; S₂: 493±436). From these results and partly also in combination with previously obtained mechanical data on intact fibres it can be concluded (1) that S₂ fibres are specialized for long-lasting force maintenance whereas S₁ fibres are adapted for slow movements; (2) intrinsic myofibrillar kinetics is not the main time-limiting factor for either activation or relaxation of intact fibres under physiological conditions; (3) processes which precede crossbridge cycling seem to be the main time-limiting factors for the Ca²⁺ activation of the myofibrils.     

Compared properties of the contractile system of skinned slow and fast rat muscle fibres

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca²⁺ threshold for activation was lower in slow than in fast fibres while Ca²⁺ concentrations required to obtain either the maximal tension or half maximal tension (pCa₅₀) were lower in fast than in slow fibres. This apparent difference in Ca²⁺ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the t _max parameter were slightly dependent on the Ca²⁺ concentration whereas the t ₅₀ parameter changed significantly with the Ca²⁺ concentration. In plantaris, both t _max and t ₅₀ parameters were found to depend strongly on the Ca²⁺ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.     

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.     

Muscle fatigue and excitation-contraction coupling responses following a session of prolonged cycling

Aim: The mechanisms underlying the fatigue that occurs in human muscle following sustained activity are thought to reside in one or more of the excitation–contraction coupling (E–C coupling) processes. This study investigated the association between the changes in select E–C coupling properties and the impairment in force generation that occurs with prolonged cycling. Methods: Ten volunteers with a peak aerobic power () of 2.95 ± 0.27 L min^?1 (mean ± SE), exercised for 2 h at 62 ± 1.3%. Quadriceps function was assessed and tissue properties (vastus lateralis) were measured prior to (E1‐pre) and following (E1‐post) exercise and on three consecutive days of recovery (R1, R2 and R3). Results: While exercise failed to depress the maximal activity (V_max) of the Na⁺,K⁺‐ATPase (P = 0.10), reductions (P < 0.05) were found at E1‐post in V_max of sarcoplasmic reticulum Ca²⁺‐ATPase (?22%), Ca²⁺‐uptake (?26%) and phase 1(?33%) and 2 (?38%) Ca²⁺‐release. Both V_max and Ca²⁺‐release (phase 2) recovered by R1, whereas Ca²⁺‐uptake and Ca²⁺‐release (phase 1) remained depressed (P < 0.05) at R1 and at R1 and R2 and possibly R3 (P < 0.06) respectively. Compared with E1‐pre, fatigue was observed (P < 0.05) at 10 Hz electrical stimulation at E1‐post (?56%), which persisted throughout recovery. The exercise increased (P < 0.05) overall content of the Na⁺,K⁺‐ATPase (R1, R2 and R3) and the isoforms β2 (R1, R2 and R3) and β3 (R3), but not β1 or the α‐isoforms (α1, α2 and α3). Conclusion: These results suggest a possible direct role for Ca²⁺‐release in fatigue and demonstrate a single exercise session can induce overlapping perturbations and adaptations (particularly to the Na⁺,K⁺‐ATPase).     

Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pigTaenia coli

Force-velocity relations, rate of ATP turnover (J_ATP), and phosphorylation of the 20,000 D myosin light chains (LC₂₀) were measured in chemically skinned guinea pigTaenia coli. Relative LC₂₀ phosphorylation at 3.2 mM MgATP was 17% in relaxed tissues at pCa 9, and increased with force at increasing [Ca²⁺] to a maximum of 67% at pCa 4.5. Force at pCa 4.5 was dependent on the MgATP concentration with a half-maximal response at about 0.1 mM. At 0.1 mM MgATP LC₂₀ phosphorylation at pCa 4.5 was 38%. Both J_ATP and the maximal shortening velocity (V _max) were reduced in 0.1 mM MgATP, to 32% and 43%, respectively, of their values at 3.2 mM MgATP. Low-MgATP thus inhibits both LC₂₀ phosphorylation and the extent and rate of cross-bridge interaction. High levels of LC₂₀ phosphorylation, independent of Ca²⁺ and MgATP concentrations, were obtained by treatment with ATP--S. Maximal force at 3.2 mM MgATP after LC₂₀ thiophosphorylation was unchanged, whereas halfmaximal force occurred at 0.065 mM MgATP after thiophosphrylation, compared to 0.13 mM after activation by Ca²⁺. The contraction in thiophosphorylated preparations at low-MgATP (0.1 mM) was associated with submaximalV _max (60%) and J_ATP (27%). The results show that LC₂₀ phosphorylation is correlated with the degree of force development in the Ca²⁺ activated contraction, both when Ca²⁺ and MgATP concentrations are varied. The reduced force and rate of crossbridge turnover in lowMgATP are however primarily mediated by an influence of MgATP on the cross-bridge cycle, which is separate from the effect on LC₂₀ phosphorylation.     

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.     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

Additive positive inotropic effects of milrinone,ouabain and calcium in diseased human ventricular myocardium

Summary The interactions of milrinone, ouabain and calcium on force of contraction in isolated, contracting human papillary muscle strips were measured. Milrinone (EC₅₀, 8 × 10^–5 M) increased force of contraction maximally by 2.8±0.8 mN at 5 × 10^–4M; significantly less than either ouabain (1 × 10^–7M; 4.8±0.5 mN increase) or calcium (15 mM; 6.2±0.6 mN increase). A submaximal, but not a maximal, inotropic effect of ouabain could be increased by the addition of milrinone; in contrast, both ouabain and calcium increased the maximal inotropic effect of milrinone by 1.7±0.2 mN and 2.7±0.3 mN, respectively. The combined inotropic effect of milrinone with either ouabain of 4.2±0.3 mN or calcium of 5.6±0.4 mN was not different from that with calcium or ouabain alone. We conclude that further positive inotropic effects should be expected when digitalis is given to patients with congestive heart failure who are already optimally treated with milrinone.Abbreviations K _D dissociation constant - (Na⁺ + K⁺)-ATPase sodium and potassium activated adenosine triphosphatase (EC 3.6.1.3) This study was carried out using human heart samples provided by: Prof. E. Kreuzer, Prof. B. Kemkes, Dr. C. Weinhold and their colleagues, Herzchirurgische Klinik der Universität, Klinikum Großhadern, München     

Activation of skinned arthropod muscle fibres by Ca2+ and Sr2+

Summary Mechanically skinned skeletal muscle fibres of three crustaceans (barnacle, crayfish and crab) and two insects (cockroach and cricket) were activated in Ca²⁺- and Sr²⁺-buffered solutions of different concentrations and the isometric force response was determined. The maximum force response induced by Sr²⁺ (P ₀ ^Sr ) was only 0–10% of that induced by Ca²⁺ (P ₀ ^Ca ) in all crustacean muscles, but approached 90% in insects. Experiments on barnacle muscle fibres activated simultaneously by Ca²⁺ and Sr²⁺ suggested that Sr²⁺ competes with Ca²⁺ for binding onto the regulatory sites without, however, being able to turn all of them on as efficiently as Ca²⁺. Interestingly, the ratioP ₀ ^Sr /P ₀ ^Ca and the sensitivity for both Sr²⁺ and Ca²⁺ increased substantially after 4–6 h following the dissection of the animals in most intact decapod muscle fibres and after 24 h in most barnacle muscle fibres. The steepness of the activation curves for both Ca²⁺ and Sr²⁺ was similar for each muscle regardless of the age of the fibre and implied that more than 2 Ca²⁺ (2 Sr²⁺) were involved in the activation process of each muscle.A Ca²⁺-induced Ca²⁺ release mechanism of physiological importance was found to operate in all arthropod muscle fibres investigated.     

Inhibition of SERCA2 Ca2+-ATPases by Cs+

Replacement of K⁺ with Cs⁺ on the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane reduces the maximum velocity (V_max) of Ca²⁺ uptake into the SR of saponin-permeabilized rat ventricular myocytes. To compare the sensitivity of the cardiac and smooth muscle/non-muscle forms of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a and -2b respectively) to replacement of K⁺ with Cs⁺, SERCA2a and SERCA2b were expressed in HEK-293 cells. Ca²⁺ uptake into HEK cell microsomes was inhibited by replacement of extravesicular K⁺ with Cs⁺ (V_max of SERCA2a-mediated Ca²⁺ uptake in CsCl was 80% of that in KCl; V_max of SERCA2b-mediated uptake was 70% of that in KCl). The Ca²⁺ sensitivity of uptake was decreased for both SERCA2a- and SERCA2b-mediated uptake and the Hill coefficients were increased in the presence of CsCl. The effects of Cs⁺ on uptake were associated with direct inhibition of the ATPase activity of SERCA2a and SERCA2b. Our results indicate that cation binding sites are present in both SERCA2 isoforms, although the extent to which SERCA2b is inhibited by K⁺ replacement is greater than that of SERCA2a or SERCA1. Consideration of these results and the recent molecular modeling work of others suggests that monovalent cations could interact with the Ca²⁺ binding region of SERCA.     

Ca2+- and Sr2+-activation properties of muscle fibres from a muscle receptor organ and the associated extrafusal muscle of the crab and crayfish

In this study on decapod crustaceans, we examined the Ca²⁺- and Sr²⁺-activation properties of skeletal muscle fibres from an identified proprioceptor, the thoracic coxal muscle receptor organ (TCMRO) and its extrafusal promotor muscle fibres. Proprioceptors and extrafusal muscles were isolated from a walking leg from the crayfish (Cherax destructor) and the rear swimming leg of the mud crab (Scylla serrata). The crayfish and mud crab TCMROs had very low Hill coefficient (n _Ca) values (1.86 ± 0.08 and 1.64 ± 0.03, respectively). In comparison to other skeletal muscle fibre types these low Hill coefficients would enable the length of the receptor muscles to be finely controlled over a wide range of [Ca²⁺]. Maximum force was found to be significantly lower in the TCMROs (crayfish: 5.76 ± 0.98; crab: 4.80 ± 0.56 Ncm^–2), compared to their associated extrafusal promotor muscle fibres (crayfish: 10.69 ± 1.63; crab: 20.07 ± 1.98 Ncm^–2), which is consistent with their sensory role. The muscle fibres of the crayfish TCMRO had faster contractile properties than the mud crab TCMRO, we discuss how these contractile properties relate to the type of locomotion undergone by each leg. The mud crab red promotor and all crayfish promotor fibres were characterised as slow with low Hill coefficients (n _Ca: crayfish: 3.22 ± 0.29; crab: 3.34 ± 0.29) and a contractile apparatus with a high sensitivity to Ca²⁺ (pCa₅₀: crayfish: 6.42 ± 0.03; crab: 6.18 ± 0.03). In contrast the white mud crab promotor fibres from the swimming leg had contractile properties that were characteristic of fast fibres with a high mean Hill coefficient (n _Ca: 5.27 ± 0.76) and a lower Ca²⁺ sensitivity (pCa₅₀: 6.03 ± 0.03). The sensitivity of the contractile apparatus to Sr²⁺ was very low (range of mean pSr₅₀: 4.23 ± 0.03–3.48 ± 0.06) and low force levels were produced in comparison to that produced with Ca²⁺. The results of this study show that the muscle fibres of the sensory receptor, produce less force and have been adapted to enable the length of the receptor to be finely set in relation to the length of the extrafusal muscle. We discuss how the striated fibres of the receptor have been adapted to perform a sensory role and how this is related to the type of locomotion undergone by the legs. We also discuss how the fibre types of the extrafusal muscle have adapted to the mode of locomotion.