Action potential parameters affecting excitation-contraction coupling

In quantifying type B potentiation effects, given earlier merely qualitatively, it is found that Zn²⁺, 1—50 µM, causes increases in action potential duration, twitch tension, and twitch contraction period time, which are all directly proportional to the log of the concentration. Hence, the duration of the action potential, i.e. the magnitude of its mechanically effective period, is a causal factor quantitatively determining the degree of mechanical activation in the isometric twitch. In higher concentrations of Zn²⁺ up to 1000 µM, the spike duration and the contraction time continue to increase but the twitch tension is disproportionately smaller, evidently because the high zinc (500—1000 µM) raises the mechanical threshold of excitation-contraction (E—C) coupling and reduces the intrinsic strength of the contractile system. Eserine (1.5 mM) and also high Zn²⁺ not only cause type B potentiation effects, but also slow the rise of the spike, thus causing retardation of the very onset of tension production, which is even greater for high Zn²⁺ because of the raised mechanical threshold. This retardation is then succeeded by the faster tension output characteristic of type B potentiation resulting from spike prolongation. Thus, the changes in the consecutive, rising and falling phases of the action potential explicitly register their separate effects in the respective very earliest and directly following periods of twitch output; i.e., each phase of the action potential produces its own mechanical "transform." These transforms, and other effects, suggest that the release of activator Ca²⁺ from the sarcoplasmic reticulum during E—C coupling can be graded in both the rate and the total amount of the release.     

Mechanical threshold as a factor in excitation-contraction coupling

I^-, CH₃SO₄ ^-, and ClO₄ ^-, like other previously studied type A twitch potentiators (Br^-, NO₃ ^-, SCN^-, and caffeine), lower the mechanical threshold in K depolarization contractures of frog skeletal muscle. In potentiated twitches, I^-, Br^-, CH₃SO₄ ^-, ClO₄, and SCN, as already reported for NO₃ ^- and caffeine, slightly shorten the latent period (L) and considerably increase the rate of tension development (dP/dt) during the first few milliseconds of the contraction period. Divalent cations (8 mM Ca²⁺, 0.5–1.0 mM Zn²⁺ and Cd²⁺) raise the mechanical threshold of contractures, and correspondingly affect the twitch by depressing the tension output, increasing L, and decreasing the early dP/dt, thus acting oppositely to the type A potentiators. These various results form a broad, consistent pattern indicating that electromechanical coupling in the twitch is conditioned by a mechanical threshold as it is in the contracture, and suggesting that the lower the threshold, in reference to the raised threshold under the action of the divalent cations, the more effective is a given action potential in activating the twitch as regards especially both its early rate and peak magnitude of tension development. The results suggest that the direct action by which the various agents affect the level of the mechanical threshold involves effects on E-C coupling processes of the T tubular and/or the sarcoplasmic reticulum which control the release of Ca for activating contraction.     

Quinine and caffeine effects on 45Ca movements in frog sartorius muscle

1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of ⁴⁵Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of ⁴⁵Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on ⁴⁵Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of ⁴⁵Ca by normally polarized muscle. However, there are variable effects of quinine upon ⁴⁵Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release ⁴⁵Ca from muscles that have been washed in Ringer''s solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca²⁺ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K⁺ or an action potential. In respect to the myoplasmic Ca²⁺ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Amino Acid and Sugar Transport in Rabbit Ileum

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10^-5 M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.     

Calcium-Binding Properties of Sarcoplasmic Reticulum As Influenced by ATP,Caffeine, Quinine,and Local Anesthetics

Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10^-5 - 10^-4 M ATP to exchange with ⁴⁵Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10^-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on ⁴⁵Ca fluxes and on the twitch output observed in whole muscles.     

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.     

Cation regulation in the smooth muscle of frog stomach

Cation composition of frog smooth muscle cells was investigated. Fresh stomach muscle rings resembled skeletal muscle, but marked Na gain and K loss followed immersion. Mean Na (49.8–79.7 mM/kg tissue) and K (61.8–80.1 mM/kg tissue) varied between batches, but were stable for long periods in vitro. Exchange of 6–30 mM Na/kg tissue with ²²Na was extremely slow and distinct. Extracellular water was estimated from sucrose-¹⁴C uptake. Calculated exchangeable intracellular Na was 9 mM/kg cell water, and varied little. Thus steady-state transmembrane cation gradients appeared to be steep. K-free solution had only slight effects. Ouabain (10^-4 M) caused marked Na gain and reciprocal K loss; at 30°C, Na and K varied linearly with time over a wide range of contents, indicating constant net fluxes. Net fluxes decreased with temperature decrease. ²²Na exchange in ouabain-treated tissue at 20–30°C was rapid and difficult to analyze. The best minimum estimates of unidirectional Na fluxes at 30°C were 10–12 times the constant net flux; constant pump efflux may explain these findings. The rapidity of Na exchange may not reflect very high permeability, but it does require a high rate of transport work.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Sodium fluxes in internally dialyzed squid axons

The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.¹ the compound d-ATP supported 25–50% of the normal sodium extrusion, while ATP supported 80–100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 µM and more gradually in the range 10 to 10,000 µM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 µM) changed the sodium efflux to about 8–12 pmoles/cm² sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.     

Effects of the Intracellular Ca Ion Concentration upon the Excitability of the Muscle Fiber Membrane of a Barnacle

The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca⁺⁺ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca⁺⁺ concentrations below about 8 x 10^-8 M, oscillatory potential changes in the range between 8 x 10^-8 to 5 x 10^-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10^-7 M. All-or-none spike potentials were suppressed when the internal Mg⁺⁺ concentration exceeded 5 mM. The suppression threshold of the internal Ca⁺⁺ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca⁺⁺ for contraction was about 8 x 10^-7 M.     

The Dual Effect of Lithium Ions on Sodium Efflux in Skeletal Muscle

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer''s solution containing 60 mM LiCl and 50 mM NaCl at 20°C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10^-5 M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer''s solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10^-5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer''s solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.     

Choline permeability in cardiac muscle cells of the cat

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm²·sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16–45 pmoles/cm²·sec in 137 mM choline and at 1.3–3.5 pmoles/cm²·sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

Effects of caffeine on crayfish muscle fibers. I. Activation of contraction and induction of Ca spike electrogenesis

Contractions are evoked in single muscle fibers of crayfish by intracellular as well as extracellular applications of caffeine. Responses to external applications in concentrations above 2 mM could be induced indefinitely. With concentrations above 5 mM the caffeine-induced responses were highly repeatable. Tensions were transient even when the caffeine remained in the bath. There was no change in resting potential, but during the contraction the effective resistance decreased about 10%. A number of factors (change in pH, Ca, K, and Cl) modified the responses. The time course of the tension was greatly prolonged when the transverse tubular system (TTS) was s swollen and was again shortened when the TTS was caused to shrink. An increased permeability to Ca induced by caffeine was evidenced by the transformation of the normally graded electrical responses to Ca spikes, which are insensitive to tetrodotoxin. The overshoot is a function of both external Ca and caffeine. A 10-fold change in Ca changed the overshoot by 19 mv in the presence of 10 mM caffeine and by 29 mv in 80 mM caffeine. The role of the increased permeability to Ca for caffeine-induced contractions will be analyzed in the accompanying paper.     

The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum

At concentrations between 1 to 10 mM, caffeine reduced the Ca-accumulating capacity of fragmented reticulum obtained from frog and rabbit muscle. With 8 mM caffeine enough Ca was released from frog reticulum to account for the force of the contracture. Caffeine did not affect all reticulum membranes equally. The fraction which was spun down at 2000 g was more sensitive than the lighter fractions. The percentage of the total accumulated Ca released by caffeine decreased with decreasing Ca content of the reticulum. In parallel with their known effects on the caffeine contracture, a drop in temperature increased the caffeine-induced Ca release while procaine inhibited it. Caffeine also inhibited the rate of Ca uptake, which may in part account for the prolongation of the active state caused by caffeine.     

Electrical properties and excitation-contraction coupling in skeletal muscle treated with ethylene glycol

The contractility of the frog sartorius muscle was suppressed after treatment with a Ringer solution added with ethylene glycol (EGR). No contraction was elicited by nerve stimulation when the muscle was brought back to normal Ringer solution after having been soaked in 876 mM EGR for 4 hr or in 1095 mM EGR for 2 hr. However, the action potential of normal amplitude was generated and followed by a depolarizing afterpotential. The resting membrane potential was slightly decreased from the mean normal value of –91.1 mv to –78.8 mv when 1095 mM EGR was used, and to –82.3 mv when 876 mM EGR was used, but remained almost constant for as long as 2 hr. The afterpotential that follows a train of impulses and a slow change in membrane potential produced by a step hyperpolarizing current (so-called "creep") were suppressed after treatment with ethylene glycol. The specific membrane capacity decreased to about 50% of the control values while the specific membrane resistance increased to about twice the control values Therefore, the membrane time constant remained essentially unchanged. The water content of the muscle decreased by about 30% during a 2 hr immersion in 1095 mM EGR, and increased by about 30% beyond the original control level after bringing the muscle back to normal Ringer. The intracellular potassium content did not change significantly during these procedures. Some differences between the present results and those obtained with glycerol are discussed.     

The Effect of Quinidine on Calcium Accumulation by Isolated Sarcoplasmic Reticulum of Skeletal and Cardiac Muscle

Quinidine potentiates twitch tension and (at higher concentrations) causes contracture of skeletal muscle whereas the same drug reduces tension development of cardiac muscle. To gain insight into the possible differences in the excitation-contraction coupling mechanism of the two types of muscle the effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum from skeletal and cardiac muscle was investigated. In a medium containing ATP, Mg⁺⁺, oxalate, and ⁴⁵Ca, pharmacologically active concentrations of the drug inhibited calcium accumulation by both skeletal and cardiac sarcoplasmic reticulum. The inhibition of the rates of calcium, uptake by the skeletal muscle preparation ranged from 11% with 10^-4 M quinidine to 90% with 10^-3 M quinidine. With the cardiac muscle preparation the inhibition ranged from 16% with 3 x 10^-6 M quinidine to 100% with 10^-3 M quinidine. With both preparations the inhibition of calcium transport was accompanied by an inhibition of the Ca⁺⁺-activated ATPase activity of the sarcoplasmic reticulum. The effect of quinidine on the skeletal sarcoplasmic reticulum supports the hypothesis that this compound produces twitch potentiation and contracture by interfering with intracellular calcium, sequestration. Its effect on cardiac sarcoplasmic reticulum. has been interpreted in terms of the hypothesis that cardiac contractility is a function of the amount of calcium released from the sarcoplasmic reticulum which is in turn dependent upon the absolute calcium content of the reticulum. Hence, following inhibition of calcium transport there would be less calcium available for coupling.