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.     

Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments

Summary It is well established that caldesmon binds to actin (K _b–10⁷-10⁸ m ^–1) and to tropomyosin (K _b10⁶ m ^–1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 10⁶ m ^–1 in low salt buffer, but is weakened by increasing [KCl] reaching 10⁵ mM^–1 in 100mm KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg²⁺ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen frayed from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous whiskers are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3mm MgATP shows there is a high affinity binding (K_b=10⁶ m ^–1) which is independent of [Ca²⁺] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the contractile domain in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought to be able to crosslink thick and thin filamentsin vivo.     

Functional effects of LC1-reassociation with cardiac papain Mg · S1

Summary The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg²⁺. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg · S1. Mixing a 10–15 m excess of isolated light chains with Mg · S1 in the presence of 10 mm ATP, 12 mm MgCl₂, 4.7 m NH₄Cl allowed LC1 to recombine with LC1-deficient Mg · S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg · S1 had a V_m of 0.4 s^-1, K_a of 30 m and a K_bind of 28 m. In the presence of intact LC1, V_m rose to 0.8 s^-1 while K_a and K_bind were reduced to 7.5 and 12 m, respectively. The fourfold decrease in K_a strongly indicated an increased affinity for actin by Mg · S1 in the presence of uncleaved LC1. Also, Ca²⁺-regulation of dog heart myofibrils was suppressed when Ca²⁺-activated MgATPase assays, as a function of Ca²⁺, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca²⁺-regulation.     

Freeze-fracture study of the mechanoreceptive digital corpuscles of mice

Summary The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 ± 517 m^–2 (mean ±s.e.m.) in the P-face and 84 ± 4 m^–2 in the E-face. Particles were 10 ± 1.8 nm in diameter in the P-face and 10 ± 1.5 nm (mean ±s.d.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 ± 224 m^–2 in the P-face and 265 ± 95 m^–2 in the E-face. Particles were 10 ± 1.4 nm in diameter in the P-face and 10 ± 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 ± 283 m^–2 and 1514 ± 514 m^–2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 ± 1.2 nm and 10 ± 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively.The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.     

Effect of phalloidin on the ATPase activity of striated muscle myofibrils

Summary Phalloidin was shown to increase the ATPase activity and Ca²⁺ sensitivity of both bovine cardiac and rabbit psoas myofibrils when assayed in a solution containing 50 mm KCl, 100 mm MOPS (pH 7.0), 2 mm MgCl₂, 1 mm ATP, 2 mm EGTA, and varying concentrations of Ca²⁺ (temperature 21–22°C). The phalloidin effect in cardiac myofibrils developed over a time course of several minutes in the presence of 50 m phalloidin. Relative increase of ATPase activity was maximal at pCa 8 and decreased with decrease in pCa. In cardiac myofibrils the increase was about 70% at pCa 8 and 20% at pCa 4 following 20–30 min pre-incubation with 2 m or 50 m phalloidin. The effect persisted after excess phalloidin was washed out. The increase in Ca²⁺ sensitivity was approximately 0.15 pCa units. For skeletal myofibrils treated with 2 m phalloidin all changes were considerably less than those seen with cardiac myofibrils and the changes were even less when the myofibrils were exposed to 50 m phalloidin. These results show that when specifically bound to actin, phalloidin can change the kinetic parameters of the cross-bridge cycle and may also alter the Ca²⁺ sensitivity of the contractile system. The effects of phalloidin seem to vary with muscle type.     

Characterization of the ATP-inhibited K+ current in canine coronary smooth muscle cells

Intracellular adenosine triphosphate (ATP)-inhibited K⁺ currents (I _{K, ATP} ) in canine coronary artery smooth muscle cells were characterized in the wholecell configuration using the suction pipette method. Cells dialysed internally with solutions containing 5 mM ATP (ATP_i) showed little detectable whole-cell current at potentials more negative than –30 mV. However, cells dialysed with ATP_i-free solutions developed a time- and voltage-independent current which reached a maximum of 132±25 pA at –40 mV about 10 min following patch rupture. After run-up, the current showed little run-down. Concentration-dependent inhibition by ATP_i yielded an inhibition constant (K _i of 350 M and a Hill coefficient of 2.3. In ATP_i-free solutions, the large current at –40 mV was reduced by glibenclamide with aK _i of 20 nM and a Hill coefficient of 0.95. Conversely, in 1 mM ATP_i solutions, the small current at –40 mV was increased by P-1075 from 8±2 pA to 143±33 pA, with a dissociation constant (K _d) of 0.16 M and a Hill coefficient of 1.7. The effect of P-1075 was antagonized by glibenclamide. Maximal current density elicited by either ATP_i depletion or external application of the channel opener P-1075 was similar with slope conductances of 81±10 pS/pF and 76±13 pS/pF respectively in the potential range of –90 to –40 mV. External Ca²⁺ had no effect on this current. Finally, in 1 mM ATP_i, 20 and 50 M adenosine increased the current slope conductance by 36±15% and 73±10% respectively between –90 to –40 mV. TheI _{K, ATP} although very small in these cells, was extremely effective in causing membrane potential hyperpolarization.     

N-Acetyl-l-cysteine and its derivatives activate a Cl− conductance in epithelial cells

N-Acetyl-l-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (we) conductance (G _m) and voltage (V _m) of NAC and the congeners S-carboxymethyl-l-cysteine (CMC) andS-carbamyl-L-cysteine (CAC) andL-cysteine in normal and CF airway epithelial cells.L-Cysteine (1 mmol/1) had no detectable effect. The increase inG _m (G_m) by the other compounds was concentration dependent and was (all substances at 1 mmol/1) 3.8 ± 1.4 nS (NAC; n = 11), 4.2 ± 1.0 nS (CMC;n = 16) and 3.8 ± 1.6 nS (CAC;n = 18), respectively. The changes in G_m were paralleled by an increased depolarization (V_m) when extracellular Cl^– concentration was reduced to 34 mmol/1: under control conditions = -4.1 ± 2.1 versus 10.2 ± 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl^– concentration was paralleled by a reduction ofG _m by 2.1 ± 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl^– conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/1). A Cl^– conductance was also activated in HT₂₉ colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPA1 cells, which do not express detectable levels of F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl^– currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-l-methylxanthine (IBMX) (Gm = 0.08 ±0.04 S;n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected cocytesG _m was enhanced when intracellular adenosine 3,5-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (G _m = 4.5 ± 1.3 S;n = 8).G _m was significantly increased by 0.74 ± 0.2 S (n = 11) and 0.46 ± 0.1 S (n = 10) when oocytes were exposed to NAC and CMC, respectively (both I mmol/1). In conclusion, NAC and its congeners activate Cl^– conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.     

A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using μg quantities of protein

Summary The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is 1 m at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a K_d below 0.1 m. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for K_d's as low as 10 nm and we demonstrate that the K_d's can be estimated using only g quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.     

Role of myofibrillar creatine kinase in the relaxation of rigor tension in skinned cardiac muscle

In the absence of creatine phosphate, MgATP produced relaxation of rigor tension in chemically-skinned right papillary muscles of the rat, the half maximal effect being obtained at 1.8 mM MgATP. In the presence of 12 mM creatine phosphate and 250 M ADP, a decrease in MgATP concentration even to 10^–9 M never induced rigor tension. At a very low MgATP concentration (10^–6 M), the half maximal relaxing effect was obtained with 2 mM creatine phosphate, a value close to theK _m of isolated MM-creatine kinase for this substrate, or with 14 M MgADP, a value 5 times lower than the reportedK _m. An exogenous MgATP regenerating system (phosphoenol pyruvate + pyruvate kinase) was not able to fully relax the fibres. When MM-creatine kinase was inhibited by fluorodinitrobenzene, the dependency of rigor tension on MgATP became the same as it was without creatine phosphate. After washing out the fluorodinitrobenzene the addition of exogenous MM-creatine kinase for half an hour fully relaxed rigor tension; moreover, this effect persisted even after prolonged washout. These results show that endogenous MM-creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a localized high MgATP/MgADP ratio; they also suggest the existence of rapidly exchangeable binding sites for MM-creatine kinase in cardiac myofibrils.     

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.     

Ca2+- and swelling-induced activation of ion conductances in bronchial epithelial cells

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of –36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl^– conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 mol/l) and ATP (0.1–100 mol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 mol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 mol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ATP > ITP > GTP CTP [,-methylene] ATP 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl^– and K⁺ conductances. The Cl^– conductance was only slightly inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl^– channels, the Ca²⁺-induced Cl^– secretion is due mainly to activation of basolateral K⁺ channels.     

Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres

Summary The effects of amrinone were studied on single skinned fibres isolated from rat hindlimb muscles. In each fibre a force-velocity relation was determined during maximal calcium activation (pCa=4.45) in control conditions and in the presence of amrinone. The MgATP concentration was 3.93 mm, close to the physiological value. After the experiment the fibre was classified as fast or slow on the basis of its reactivity with anti-myosin monoclonal antibodies. In fast fibres amrinone (3 mm) potentiated isometric tension (P ₀) by 13.8±2.9% (n=13), reduced maximum shortening velocity (V _max ) by 32.6±3.2% and the curvature of the force-velocity relation (a/P ₀) was increased by 98.9±46.0%. All these effects were less pronounced in slow fibres, where V _max was reduced only by 11.4±3.6 (n=16). The effects of amrinone (0.3–6 mm) on the ATPase activity of myofibrils and myosin prepared from fast (tibialis anterior) and slow (soleus) rat skeletal muscles were studied. Amrinone was found to depress Ca–Mg dependent ATPase activity of myofibrillar preparations of the tibialis anterior (up to 16.6±2%) and, to a lesser extent, of the soleus (up to 7.2±1.2%). On the contrary, Ca-stimulated myosin ATPase activity was significantly increased by amrinone in myosin preparations from the tibialis anterior. Experiments were carried out to test whether amrinone (3 mm) might affect the sensitivity of the contractile system to MgATP concentration ([MgATP]). The results obtained showed that (1) the [MgATP] value at which isometric tension reached its maximum was shifted by amrinone from 0.1 mm to 0.3 mm, (2) the slope of the negative relation between [MgATP] and a/P ₀ was made more steep by amrinone, and (3) the Km of the hyperbolic relation between [MgATP] and V _max was increased from 0.39 to 1.71 mm by amrinone, thus indicating a reduced affinity of myosin for MgATP. These results are in accordance with the hypothesis that amrinone exerts a direct effect on the contractile mechanism.     

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres

Summary The deteriorating effect of m order of Ca²⁺ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins by calcium-activated neutral protease (CANP). Tension developed in solutions containing no MgATP (rigor solution) decreased irreversibly with the addition of Ca²⁺ in quantities of more than 0.1 m. Low temperature was seen to suppress (Q₁₀>4), and neutral pH to maximize, this decrease in tension. In rigor solution containing Ca²⁺, SDS electrophoresis indicated that a 95 k dalton component (-actinin) was released from the fibre; electron micrography showed the disappearance of Z-lines. These results suggest that one of the causes for decrease in rigor tension is the proteolytic activity of CANP, and its inhibitors were shown to be quite useful in experiments on skinned fibre.     

Stimulus-specific patterns of myosin light chain phosphorylation in smooth muscle of rabbit thoracic artery

When the rabbit thoracic artery was stimulated with submaximal concentrations of agonist [40 mM K⁺, 30 M prostaglandin F₂ (PGF₂) or 7 M histamine], about 90% of a maximal contraction occurred. Each agonist induced a rapid development of contraction followed by a sustained response. The maximal rate of force generation stimulated with PGF₂ was twice that seen with K⁺ or histamine. Stimulation with 40 mM K⁺ increased the extent of monophosphorylated 20 kDa myosin light chain (MLC-P) for up to 1 min to a maximal value of 38.8±1.0%, there was a subsequent rapid decrease and the MLC-P level remained just above the basal value for 40 min (6.8±3.0%). In the case of stimulation with 7 M histamine, MLC-P level increased rapidly and was sustained for up to 40 min (28.0±4.9%). In contrast to the stimulation with K⁺ or histamine, PGF₂ induced both mono- and diphosphorylated MLC₂₀ (MLC-P and MLC-P 2 respectively) at a low concentration (3 M). The monophosphorylation of MLC₂₀ induced by 30 M PGF₂ reached the maximal value of 32.8±5.2%, and was sustained for up to 40 min (15.2±5.4%). The diphosphorylation of MLC₂₀ increased rapidly (7.4±4.0% at 5 min), then decreased to the basal value within 40 min. These results suggest that different modes of stimulation of smooth muscle contraction produce different profiles of MLC₂₀ phosphorylation. The implications of these observations are that the diphosphorylated form, specifically induced by certain agents, may modify the mode of contraction of the aortic artery.     

Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine

Inward Na⁺ currents associated with the cloned intestinal Na⁺/glucose cotransporter expressed in Xenopus oocytes have been studied using the two-microelectrode voltage-clamp method. The steady-state current/voltage relations showed voltage-dependent (V _m from +20 to –75 mV) and relatively voltage-independent (V _m from –75 to –150 mV) regions. The apparent I _max for Na⁺ and glucose increased with negative membrane potentials, and the apparent K _0.5 for glucose (K _0.5 ^Glc ) depended on V _m and [Na]_o. Increasing [Na]_o from 7 to 110 mmol/l had the same effect in decreasing K _0.5 ^Glc from 0.44 to 0.03 mmol/l as increasing the V _m from –40 to –150 mV. The I/V curves under saturating conditions (20 mmol/l external sugars and 110 mmol/l [Na]_o) were identical for d-glucose, d-galactose, -methyl d-glucopyranoside and 3-O-methyl d-glucoside. The specificity of the cotransporter for sugars was: d-glucose, d-galactose, -methyl d-glucopyranoside > 3-O-methyl d-glucoside d-xylose > d-allose d-mannose. K _i for phlorizin ( 10 mol/l) was independent of V _m at saturating [Na]_o. We conclude that a variety of sugars are transported by the cloned Na⁺/glucose cotransporter at the same maximal rate and that membrane potential affects both the maximal current and the apparent K _0.5 of the cotransporter for Na⁺ and glucose.     

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.     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Active movement of cardiac myosin on Characeae actin cables

The active sliding of cardiac myosin on actin cables was studied using an in vitro movement assay. Cardiac myosin prepared from either adult rabbit or rat hearts was mixed with small latex beads to coat them. Actin cables were obtained from the internodal cells of green algae, Characeae. When the myosin-coated beads suspended in physiological buffer were introduced into the internodal cells, the myosin started to interact with the actin causing the beads to move. The sliding movement of the beads was observed under microscopy and the sliding velocity measured. The observed movement was smooth and the velocity was constant over a long distance. The movement was physiological in nature: a) it was ATP-dependent, but above a certain level of ATP, the velocity was constant; b) the velocity was maximum at pH 7.0, and decreased in both acidic and alkaline conditions. The average sliding velocity of cardiac myosin obtained from rabbit ventricles (0.31±0.11 m/s) was slower than that from rat ventricles (1.04±0.26 m/s) reflecting the lower ATPase activity of rabbit cardiac myosin. This assay system is considered to be a useful tool linking biochemistry and physiology at the molecular level.Supported in part by a Mitsukoshi Medical Award, Tokyo, Japan     

Cytoplasm-to-myonucleus ratios in plantaris and soleus muscle fibres following hindlimb suspension

Summary In multinucleated skeletal muscle fibres the size of the cytoplasmic volume-to-myonucleus ratio is related to the myosin heavy chain phenotype, with the ratio being larger in those fibres expressing the fast myosin heavy chain phenotype. It is unknown, however, whether this ratio is modulated during muscle fibre adaptation, such as that which occurs following muscle unloading. In this study the relationship between cross sectional area, myonuclear number and myosin type, in single fibres from the plantaris and soleus muscles of adult rats following 28 days of hindlimb suspension was examined. Each fibre was cut transversely into two segments; one segment was used for immunohistochemical identification of myosin type, the other for determination of cross sectional area and myonuclei number. Single fibre analysis revealed significant atrophy of both plantaris fast and soleus slow fibres; the mean cross sectional area (m²) of these fibres, 3104±183 and 2082±107 (mean ±se), being 70 and 45%, respectively, of control means. The decreases in cross sectional area were not accompained by corresponding decreases in the number of myonuclei (myonuclei/mm); in plantaris fast fibres the mean myonuclei counts were within the control range (88±8 (hindlimb suspension), 76±7 (control)), in soleus slow fibres the counts were significantly increased (185±12 (hindlimb suspension), 154±11 (control)). The changes resulted in a significant decrease in the cytoplasmic volume-to-myonucleus ratio (m³×10³) for both fibre types; the mean ratios of 39±3 and 12±1, were 60% and 36% of control means for the plantaris fast and soleus slow fibres, respectively. These results indicate that following hindlimb suspension atrophy of muscle fibres the myonuclei numbers remain constant or increase and, hence, the effective cytoplasmic-to-myonucleus ratio is decreased. Further, the described changes are significantly greater in soleus slow than plantaris fast fibres.