Myonemal contraction of Spirostomum. I. Kinetics of contraction and relaxation

A microphotometric method is introduced that allows measurement of the contraction-relaxation kinetics of Spirostomum in response to electrical stimulation. The time course of contraction includes a rapidly contracting phase of some 4–5 mS during which cells shorten at a rate in excess of 100 cell lengths sec^?1. While a stimulus strength-duration curve determines the threshold of the response, the response to above threshold stimuli of different strengths and to trains of stimuli suggest that contraction of Spirostomum may not be an all-or-none event. The kinetics of relaxation following high stimulating voltages and repetitive after contractions also induced by high voltages are explained by excitation-contraction coupling through a stimulus-dependent intermediate effector, possibly the release of calcium ions. Changes in resting membrane potential detected by intracellular recording do not influence the initiation of contraction, while microinjection of calcium buffers above 10^?5 M Ca²⁺ invariably induces contraction.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Validity of the Force-Velocity Relation for Muscle Contraction in the Length Region, l ≤ l0

Considerable attention has been directed to the characteristic force-velocity relation discovered by A. V. Hill in the study of muscle kinematics. Models of contractile process were tested on the basis of their compatibility with the Hill equation. However, almost all the isotonic data have been restricted to one length, l₀, the maximum length with almost no resting tension; the velocities measured are those initial values when the load begins to move. The force-velocity curve extrapolates to zero velocity for isometric tension, but only for the tension at that one length. Very few efforts have been made to study the profiles of the curves throughout the range of lengths over which shortening takes place. In examining the length region, l ≤ l₀, for an isotonically contracting muscle, not only is the force-velocity relation valid for the initial reference length, l₀, but also for any other length. The analysis in this report indicates that the constants a/P₀ and b/l₀ remain fixed throughout the length change of afterloaded isotonic shortening in the Rana pipiens sartorius muscles.     

The Role of the Frank–Starling Law in the Transduction of Cellular Work to Whole Organ Pump Function: A Computational Modeling Analysis

We have developed a multi-scale biophysical electromechanics model of the rat left ventricle at room temperature. This model has been applied to investigate the relative roles of cellular scale length dependent regulators of tension generation on the transduction of work from the cell to whole organ pump function. Specifically, the role of the length dependent Ca²⁺ sensitivity of tension (Ca₅₀), filament overlap tension dependence, velocity dependence of tension, and tension dependent binding of Ca²⁺ to Troponin C on metrics of efficient transduction of work and stress and strain homogeneity were predicted by performing simulations in the absence of each of these feedback mechanisms. The length dependent Ca₅₀ and the filament overlap, which make up the Frank-Starling Law, were found to be the two dominant regulators of the efficient transduction of work. Analyzing the fiber velocity field in the absence of the Frank-Starling mechanisms showed that the decreased efficiency in the transduction of work in the absence of filament overlap effects was caused by increased post systolic shortening, whereas the decreased efficiency in the absence of length dependent Ca₅₀ was caused by an inversion in the regional distribution of strain.     

Activation of two types of fibres in ferret,Mustela putorius furo,cremaster muscle

Summary Some contractile, histochemical, morphological and electrophysiological properties of ferret, Mustela putorius furo, cremaster muscle have been estimated. Histochemical fibre typing revealed the presence of two types of fibres (type I 66.2%, type II 33.8%). Morphometry performed on ATPase-stained transverse sections showed that type I was composed of a large amount (40%) of small(<400 m²) cells. In mammalian Ringer two groups of fibres could be recognized on the basis of the values of resting potential (-69.7 mV and-59.1 mV) and intracellular sodium activity (8.3 mmol·l^-1 and 14.1 mmol·l^-1, respectively). In experiments on fibre bundles, the elevation of extracellular potassium concentration to 15–200 mmol·l^-1 produced contractures that consisted of a well-defined transient or phasic tension followed by a sustained or tonic tension. Properties of activation and inactivation of the tension analysed in small bundles of cut fibres (lengths 0.5–1.0 cm) were of fast- and slow-twitch type for phasic and tonic phase, respectively. In contrast to the phasic component of K contractures, the tonic phase was abolished by Ca²⁺ withdrawal and inhibited by Ni²⁺, Cd²⁺, Co²⁺, Gd³⁺ and gallopamil (D600). In Ca²⁺-free medium the sustained tension was restored by adding Sr²⁺. It is concluded that in ferret cremaster muscle the presence of slow-twitch fibres would give rise to the tonic component of the K contracture in which an extracellular source of activator Ca²⁺ is involved. The ability of these fibres to contract with a maintained tension for prolonged periods of time might participate in the temperature regulation of the testes.Abbreviations a _i ^Na intracellular sodium activity - ATPase myosin adenosine triphosphatase - D600 gallopamil - E _m membrane potential - E _r resting potential - EDL muscle, extensor digitorum longus muscle - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - e.c. excitation-contraction - SDHase succinate dehydrogenase - NADHase nicotinamide adenine, dinucleotide hydrogen-diaphorase - SOL muscle, soleus muscle - T time constant of relaxation - TEACI tetraethylammonium chloride - [Ca]_o, [K]_o, [Na]_o extracellular calcium, potassium, sodium concentration     

Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin-myosin composite filament is a dual-stage molecular spring.

The mechanical roles of sarcomere-associated cytoskeletal lattices were investigated by studying the resting tension-sarcomere length curves of mechanically skinned rabbit psoas muscle fibers over a wide range of sarcomere strain. Correlative immunoelectron microscopy of the elastic titin filaments of the endosarcomeric lattice revealed biphasic extensibility behaviors and provided a structural interpretation of the multiphasic tension-length curves. We propose that the reversible change of contour length of the extensible segment of titin between the Z line and the end of thick filaments underlies the exponential rise of resting tension. At and beyond an elastic limit near 3.8 microns, a portion of the anchored titin segment that adheres to thick filaments is released from the distal ends of thick filament. This increase in extensible length of titin results in a net length increase in the unstrained extensible segment, thereby lowering the stiffness of the fiber, lengthening the slack sarcomere length, and shifting the yield point in postyield sarcomeres. Thus, the titin-myosin composite filament behaves as a dual-stage molecular spring, consisting of an elastic connector segment for normal response and a longer latent segment that is recruited at and beyond the elastic limit of the sarcomere. Exosarcomeric intermediate filaments contribute to resting tension only above 4.5 microns. We conclude that the interlinked endo- and exosarcomeric lattices are both viscoelastic force-bearing elements. These distinct cytoskeletal lattices appear to operate over two ranges of sarcomere strains and collectively enable myofibrils to respond viscoelastically over a broad range of sarcomere and fiber lengths.     

Contraction of protoplasm. III. Cinematographic analysis of the contraction of some heterotrichs

The contraction of Stentor and Blepharisma, in response to mechanical and electrical stimulation and of Spirostomum in response to mechanical stimulation is described. All three species respond to electrical stimulation by contraction of the cytoplasm, beginning at the anodal end regardless of orientation of the animal. The differences in contractile ability and shapes during contraction are discussed in relation to body form and microanatomy. Stentor and Spirostomum also respond to mechanical stimulation. Dropping a weight on the slide causes contraction of the whole body of Spirostomum, but not of Stentor. Stimulation of the oral region of Stentor by means of a vibrating needle causes a contraction of the entire body, but this sensitivity is limited to the oral region. Blepharisma does not respond to mechanical stimulation. Spirostomum and Stentor undergo rapid spontaneous contractions, but Blepharisma does not contract spontaneously.     

The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments

Electron microscopy was used to study the positional stability of thick filaments in isometrically contracting skinned rabbit psoas muscle as a function of sarcomere length at 7 degrees C. After calcium activation at a sarcomere length of 2.6 micron, where resting stiffness is low, sarcomeres become nonuniform in length. The dispersion in sarcomere length is complete by the time maximum tension is reached. A-bands generally move from their central position and continue moving toward one of the Z-discs after tension has reached a plateau at its maximum level. The lengths of the thick and thin filaments remain constant during this movement. The extent of A-band movement during contraction depends on the final length of the individual sarcomere. After prolonged activation, all sarcomeres between 1.9 and 2.5 micron long exhibit A-bands that are adjacent to a Z-disc, with no intervening I-band. Sarcomeres 2.6 or 2.7 micron long exhibit a partial movement of A-bands. At longer sarcomere lengths, where the resting stiffness exceeds the slope of the active tension-length relation, the A-bands remain perfectly centered during contraction. Sarcomere symmetry and length uniformity are restored upon relaxation. These results indicate that the central position of the thick filaments in the resting sarcomere becomes unstable upon activation. In addition, they provide evidence that the elastic titin filaments, which join thick filaments to Z-discs, produce almost all of the resting tension in skinned rabbit psoas fibers and act to resist the movement of thick filaments away from the center of the sarcomere during contraction.     

Length-dependent deactivation of ventricular trabeculae in the bivalve, Spisula solidissima

Shortening-deactivation has been identified and characterized in ventricular trabeculae of the bivalve, Spisula solidissima (Heterodonta, Mactridae). This muscle had ultrastructural similarities to vertebrate smooth muscle. Deactivation was defined as the fraction of maximal force lost during a contraction when a muscle is shortened rapidly (by a quick-release, QR) to a known length, relative to a control isometric contraction at that same length. The magnitude of deactivation was dependent on the size of the release and the point at which the release was applied during the cycle of contraction. QR/quick-stretch (QS) perturbations at the same point during the contraction resulted in negligible deactivation. The magnitude of deactivation was independent of shortening rate. Deactivation was attenuated by applying caffeine (100 μM) and blocked with high extracellular Ca²⁺ (56 mM). The Ca²⁺ ionophore, A23187 (10 μM), augmented deactivation as did the positive inotrope serotonin (100 nM). Treatment with ryanodine (5 μM) had no significant effect on deactivation. These results suggest that a reduction in Ca²⁺ at the contractile element and/or sequestration of Ca²⁺ may occur during shortening. Deactivation may minimize the magnitude of work done during active shortening of bivalve cardiac muscle, particularly against the low afterload exhibited in the bivalve peripheral circulatory system. Intracellular Ca²⁺ fluxes during sudden length perturbations may explain the effect of stretch on action potential duration in the bivalve heart, as shown previously.     

Correlation of electrophysiological,histochemical, and mechanical properties in fibres of the coxa rotator muscle of the locust,Locusta migratoria

Summary The fibre composition of the anterior coxa rotator muscle of the locust middle leg (M92) was examined. The muscle is composed of 90–100 fibres. Muscle fibres were characterized with regard to innervation pattern, electrophysiological properties, and morphological parameters. Activity and isoenzyme composition of myofibrillar ATPase, succinic acid dehydrogenase (SDH) activity and glycogen content were examined employing histochemical techniques. Shortening velocity and the dependence of tension on intracellular Ca²⁺ were determined in skinned fibre experiments. A close match was observed between the innervation pattern of the muscle fibres and their histochemical and physiological properties. The combination of all parameters examined allowed an accurate classification of the muscle fibres into three types. Within a given type, broad variability of some properties was observed (SDH activity, Ca²⁺ sensitivity) while others assumed distinct values (innervation pattern, shortening velocity). The comprehensive characterization of muscle fibre properties permits a functional interpretation of fibre heterogeneity with regard to muscle performance. Fibres with the same innervation pattern may be recruited specifically, according to their electric properties and Ca²⁺ sensitivities. The resulting specific recruitment of fibres with different mechanical responses should allow a subtle control of muscular force, with regard to force amplitude, temporal characteristics of contraction, and metabolic cost.Abbreviations CI₁ common inhibitory neurone one - ejp ijp excitatory, inhibitory junctional potential - EGTA ethylene glycol-bis[-aminoethyl ether] N,N,N,N-tetraacetic acid - mATPase myofibrillar adenosinetriphosphatase - MOPSO 3-[N-morpholino]-2-hydroxypropanesulfonic acid - M92 anterior rotator muscle of the coxa - n Hill coefficient - pCa₅₀ pCa corresponding to half-maximal tension - P₀ maximal isometric tension - SDH succinic acid dehydrogenase - V _max maximal shortening velocity     

Studies on the origin of resting tension of skeletal muscle

Glycerol extracted frog skeletal muscle fibres at 2.2 μm sarcomere length (in situ-length) in a solution free of Ca⁺⁺ and Mg⁺⁺ but containing ATP, show a decrease in both their resting tension and their elastic modulus, if the ionic strength of the bathing solution is increased. This finding is compared with the behaviour of intact skeletal muscle fibres in hypertonic solution. It is concluded that the resting tension of intact skeletal muscle fibres at in situ-length is caused by the longitudinal sarcoplasmic reticulum as well as by interactions between the contractile filaments.     

Theoretical series elastic element length in Rana pipiens sartorius muscles

Assuming a two component system for the muscle, a series elastic element and a contractile component, the analyses of the isotonic and isometric data points were related to obtain the series elastic stiffness, dP/dl_s, from the relation, See PDF for Equation From the isometric data, dP/dt was obtained and shortening velocity, v, was a result of the isotonic experiments. Substituting (P₀ - P)/T for dP/dt and (P₀ - P)/(P + a) times b for v, dP/dl_s = (P + a) /bT, where P < P₀, and a, b are constants for any lengths l ≤ l₀ (Matsumoto, 1965). If the isometric tension and the shortening velocity are recorded for a given muscle length, l₀, although the series elastic, l_s, and the contractile component, l_c, are changing, the total muscle length, l₀ remains fixed and therefore the time constant, T. Integrating, See PDF for Equation the stress-strain relation for the series elastic element, See PDF for Equation is obtained; l_sc0 - l_s + l_c0where l_co equals the contractile component length for a muscle exerting a tension of P₀. For a given P/P₀, l_s is uniquely determined and must be the same whether on the isotonic or isometric length-tension-time curve. In fact, a locus on one surface curve can be associated with the corresponding locus on the other.     

Contractile responses in rat extensor digitorum longus muscles at different times of postnatal development

Some contractile properties of small bundles (100–200 m diameter) of muscle fibres isolated from the extensor digitorum longus muscle of rats at different times of development were compared. An increase of resting potential was observed in these muscles from-26.9 mV at 1 day of age to-72.6 mV at 3 months. Twitch tension and duration of postnatal muscles 1–7 days were diminished by reducing [Ca]_o (substituted by Mg²⁺) or adding inorganic cations (Ni²⁺, Cd²⁺, La³⁺), unlike in the oldest animals (14 days–3 months postnatal) where twitch responses were unaffected. In the latter, potentiation of the twitch tension was even recorded in the presence of Ni²⁺ (0.5–1 mmol·l^-1) and Cd²⁺ (0.5–2 mmol·l^-1). Properties of activation and inactivation of the developed tension following elevation of [K]_o to 15–200 mmol·l^-1 were analysed at the same stages of postnatal development. In contrast to the tension-membrane potential curves for activation, which presented an average negative shift of-17.6 mV between 1 day postnatal and 3 months of age, a voltage dependence of inactivation similar to that encountered in adult extensor digitorum longus muscles, was already reached at 7 days of age. These results suggest an asynchronism in the maturation of the potential-dependent characteristics of the depolarization-contraction coupling mechanism. Furthermore, during the first week postnatal, in relation with poorly developed membrane systems and low [Ca]_i-recycling capability, [Ca]_o plays a fundamental role in maintaining contraction by replenishing the intracellular calcium pool.Abbreviations ATPase adenosine triphosphatase - [Ca]_o ([K]_o) extracellular calcium (potassium) concentration - DC depolarization-contraction - EC excitation-contraction - e.d.l. muscle extensor digitorum longus muscle - E _m membrane potential - E _r resting potential - HEPES N-2 hydroxyethylpiperazine-N-2 ethanesulphonic acid - I _fast fast calcium current - sr sarcoplasmic reticulum - T-tubules transverse tubules     

大鼠离体乳头肌收缩末期张力—长度关系的曲线性

大鼠离体左室乳头肌固定于最适初长位,逐步递减“后荷”获得一系列等张收缩的张力、长度缩短程度和速度。结果发现:(1)收缩末期张力-长度关系(ESTLR)为指数曲线,回归方程 T=ar~(-bL)-K 拟合的优度明显高于线性方程拟合的优度(P<0.001),其中 a,k 分别代表总张力和静息张力,b 为曲线的弯曲度;(2)在高钙(4mmol/L)或去甲肾上腺素(NE10~(-6)mol/L)作用下,ESTLR 右上移位,a,b 和无张力缩短速度 L_O 均增大(P均<0.01),尤以高钙时的变化更明显,(3)NE 使张力-速度曲线的右上移位比高钙显著。这提示大鼠离体心肌的 ESTLR 呈非线性特征,参数 a,b 及长度轴截距 L_O 对收缩强度的变化敏感,但对收缩速度改变的敏感性可能比经典的力学指标低。     

Ca2+ protection from the negative inotropic effect of contraction frequency on teleost hearts

Summary Isometric tension development by ventricular strips of 9 species of teleosts, a frog and a turtle was assessed at varying contraction frequencies and Ca_o (external calcium concentration). With teleost hearts an increase in contraction frequency at constant Ca_o was always associated with a decrease in tension development; however, under comparable conditions a positive staircase was exhibited by the frog and turtle heart preparations. The reaction of the teleost heart was thus very different from the well established response of the hearts of higher vertebrates. Elevations in Ca_o always resulted in an increase in tension development such that the positive inotropic effect of Ca_o could compensate for the negative effect of a high contraction frequency. Perfused isolated cod hearts exhibited an increase in cardiac output and pressure development as a result of increases in Ca_o. At 30 contractions min⁻¹ a transition from 1–2 mM Ca_o led to a 68% increase in performance defined as the product of cardiac output times pressure development. The response was in excess of that of ventricular strips. At low Ca_o increases in rate from in situ resting levels to the high end of the physiological range resulted in a decrease in performance. Increases in Ca_o were able to ameliorate the detrimental effect of high imposed contraction frequency. In conclusion, both ventricular strip and perfused heart experiments show that a positive inotropic effect of increased Ca_o can compensate for or even surpass the negative effect of high contraction frequency when both variables are at physiological levels. This finding could have relevance to the maintenance of cardiac performance during/or following intense swimming when both heart rate and plasma calcium may be elevated.     

The progressive effects of a fat enriched diet on ventricular myocyte contraction and intracellular Ca2+ in the C57BL/6J mouse

The C57BL/6J mouse has a genetic susceptibility to develop diabetes when fed with a high-fat, high-sucrose diet. The general characteristics of diet-induced diabetes in this model include progressive development of hyperinsulinaemia, hyperglycaemia, insulin resistance and obesity, features that are frequently observed in the clinical setting. This study investigated the progressive effects of a fat enriched (FE) diet on contraction and intracellular Ca²⁺ in ventricular myocytes from the C57BL/6J mouse. The characteristics of the mice fed with the FE diet compared to mice receiving control diet included progressive increase in the rate of body weight gain, increased fasting blood glucose and time-dependent differences in the disposal of blood glucose after a glucose challenge. The ultrastructure of cardiac myocytes and associated capillaries did not show any gross morphological alteration after 27 weeks of FE diet compared to controls.At 5 months the resting cell length (RCL) and the kinetics of shortening were not significantly altered in ventricular myocytes from mice receiving the FE diet compared to age-matched controls. At 5 and at 7 months the amplitude of shortening was increased in myocytes receiving the FED diet compared to controls. At 7 months the time to half (THALF) relaxation of myocyte contraction was shortened in myocytes from mice receiving the FE diet compared to controls. Mean THALF relaxation in myocytes from mice fed the FE diet was 32.0 ± 1.4 ms (n = 23) compared to 40.2 ± 2.0 ms (n = 27) in controls. Neither resting intracellular Ca²⁺ nor the kinetics or amplitude of the Ca²⁺ transient were altered by FE diet. Differences in myofilament sensitivity to Ca²⁺ might underlie the changes in contractility.     

The effect of the ionophore A23187 on the ultrastructure and electrophysiological properties of frog skeletal muscle

Summary The divalent cation ionophore A23187 has three major effects on the thin cutaneous pectoris muscle of frog: (1) The membrane potential is depolarized, an action that is found only when the [Ca²⁺] of the bathing saline is very low. (2) It causes an increase in resting tension and the development of contraction. This action is produced at both normal and low values of [Ca²⁺]_o and is, therefore, independent of Ca²⁺ entry and of changes in E_m. The ionophore is believed to act primarily by releasing Ca²⁺ from intracellular stores. (3) It causes major ultrastructural damage to the muscle filaments. It is believed that this damage is the result of the action of A23187 on the sarcoplasmic reticulum and the elevation of [Ca²⁺]_i and we suggest that the action of this ionophore may serve as a useful model for the study of certain myopathies.     

Thick filament movement and isometric tension in activated skeletal muscle.

Thick filaments can move from the center of the sarcomere to the Z-disc while the isometric tension remains stable in skinned rabbit psoas fibers activated for several minutes (Horowits and Podolsky, 1987). Using the active and resting tension-length relations and the force-velocity relation, we calculated the time course and mechanical consequences of thick filament movement in the presence and absence of the elastic titin filaments, which link the ends of the thick filaments to the Z-discs and give rise to the resting tension. The calculated time course of thick filament movement exhibits a lag phase, during which the velocity and extent of movement are extremely small. This lag phase is dependent only on the properties of the cross-bridges and the initial position of the thick filament. The time course of thick filament movement in skinned rabbit psoas fibers at 7 degrees C is well fit assuming a small initial thick filament displacement away from the center of the sarcomere; this leads to a lag of approximately 80 s before any significant thick filament movement occurs. In the model incorporating titin filaments, this lag is followed by a phase of slow, steady motion during which isometric tension is stable. The model excluding titin filaments predicts a phase of acceleration accompanied by a 50% decrease in tension. The observed time course of movement and tension are consistent with the model incorporating titin filaments. The long lag phase suggests that in vivo, significant movement of thick filaments is unlikely to occur during a single contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Force-Velocity Relationship of Shortening of Blood Vessel of the Common Frog Rana temporaria during Phasic and Tonic Contractions

The relationship between shortening velocity and the applied external load (force-velocity) was studied for vena cava posterior of the frog Rana temporaria. This relationship was determined in 15 s, 2 and 20 min after the beginning of isometric contraction induced by a high KCl concentration (110 mmole/l). It was found that maximal shortening velocity (V ₀) was the highest at the 15th s after the beginning of the isometric contraction and amounted to 0.125 ± 0.009 L ₀/s (n = 4); then it decreased progressively to 0.058 ± 0.005 L ₀/s at the 20th min, respectively. On the contrary, the isometric tension value increased and reached its peak in 5 min after the beginning of contraction, then it decreased slightly. Such phenomenon indicates the occurrence of attached non-cycling bridges. The a/P ₀ parameter was equal to 0.15 ± 0.03 and 0.33 ± 0.06 at the 15th s and the 20th min, respectively.     

Calcium buffering in presynaptic nerve terminals. Free calcium levels measured with arsenazo III

The particulate fraction from osmotically shocked synaptosomes (‘synaptosomal membranes’) sequesters Ca when incubated with ATP-containing solutions. This net accumulation of Ca can reduce the free [Ca²⁺] of the bathing medium to sub-micromolar levels (measured with arsenazo III). Two distinct types of Ca sequestration site are responsible for the Ca²⁺ buffering. One site, presumed to be smooth endoplasmic reticulum, operates at low [Ca²⁺] (less than 1 μM), and has a relatively small capacity. Ca sequestration at this site is prevented by the Ca²⁺ ionophore, A-23187, but not by mitochondrial poisons. The second (mitochondrial) site, in contrast, is blocked by the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Since the intraterminal organelles can buffer [Ca²⁺] to about 0.3–0.5 μM, this may be an upper limit to the normal resting level of [Ca²⁺]_i in nerve terminals. In the steady state, total cell Ca and [Ca²⁺]_i will be governed principally by Ca transport mechanisms in the plasmalemma; the intracellular organelle transport systems then operate in equilibrium with this [Ca²⁺]. During activity, however, Ca rapidly enters the terminals and [Ca²⁺]_i rises. The intracellular buffering mechanisms then come into play and help to return [Ca²⁺]_i toward the resting level; the non-mitochondrial Ca sequestration mechanism probably plays the major role in this Ca buffering.