Frog striated muscle is permeable to hydroxide and buffer anions.

Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high K2SO4 solutions at pHo = 7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities. In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K+ was replaced by Cs+ prior to changes of the anion under study. At constant external Cs+ activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H+ permeability, calculations show that the reduction in [H+]o cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH- and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pHo = 10.0 +/- 0.3: (POH/PK) = 890 +/- 150, (PCAPS/PK) = 12 +/- 2, (PCHES/PK) = 5.3 +/- 0.9, and (PNO3/PK) = 4.7 +/- 0.5. PNO3/PK was independent of pHo up to 10.75. At pHo = 9.6, (PHCO3/PK) = 0.49 +/- 0.03; at pHo = 8.9, (PCl/PK) = 18 +/- 2 and at pHo = 7.1, (PHEPES/PK) = 20 +/- 2. In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 mM CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH- and the absorption of internal H+ by entering CAPS- anion. The rate of CAPS- entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value.(ABSTRACT TRUNCATED AT 400 WORDS)

Response of chloride efflux from skeletal muscle of Rana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment.

Efflux of 36Cl- from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl- efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. For temperatures between 0 and 20 degrees C, the measured activation energy is 7.5 kcal/mol for Cl- efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl- efflux. The pH-dependent Cl-efflux can be described by the relation mu = 1/(1 + 10n(pK alpha-pH], where mu is the Cl- efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150 mM KCl plus 120 mM NaCl (internal potential about -15 mV), the apparent pK alpha is 6.5 at both 0 and 20 degrees C, and n = 2.5 for 0 degrees C and 1.5 for 20 degrees C. For muscles equilibrated in solutions containing 7.5 mM KCl plus 120 mM NaCl (internal potential about -65 mV), the apparent pK alpha at 0 degrees C is 6.9 and n is 1.5. The voltage dependence of the apparent pK alpha suggests that the critical pH-sensitive moiety producing the pH-dependent Cl- efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact that n is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl- efflux increment on raising external pH. The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl- efflux according to the relation, efflux = exp(-k.[DEPC].t), where t is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mM). At 17 degrees C, k-1 = 188 mM . min. For temperatures between 10 and 23 degrees C, k has an apparent Q10 of 2.5. The Cl- efflux inhibitor SCN- at a concentration of 20 mM substantially retards the reduction of the pH-dependent Cl- efflux by DEPC. The findings that the apparent pK alpha is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl- efflux, and that this action is retarded by SCN- supports the notion that protonation of histidine groups associated with Cl- channels is the controlling reaction for the pH-dependent Cl- efflux.

Zinc inhibition of chloride efflux from skeletal muscle of Rana pipiens and its modification by external pH and chloride activity.

Efflux of 36Cl- from frog sartorius muscles equilibrated in depolarizing solutions was measured. Cl- efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. In depolarized muscles from Rana pipiens, the pH-dependent Cl- efflux has an apparent pKa near 6.4. The reduction of Cl- efflux by external Zn2+ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl- efflux was also examined. At pH 6.5 and a membrane potential of -22 mV, increasing external Cl- activity from 0.108 to 0.28 M decreased inhibition of the pH-dependent Cl- efflux at all activities of Zn2+. The Zn2+ activity needed to reduce Cl- efflux by half increased from 0.39 x 10(-3) to 2.09 x 10(-3) M. By contrast, external Cl- activity had no measurable effect on the apparent pKa of the pH-dependent efflux. At constant Cl- activity less than 0.21 M, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn2+ activities with either a slight increase or no change in the Zn2+ activity producing half-inhibition. In other words, for relatively low Cl- activities, protection against inhibition of Cl- efflux by low Zn2+ activities was obtained by raising, not lowering, external pH; this is not what is expected if H+ and Zn2+ ions compete at the same site to produce inhibition of Cl- efflux. We conclude that Zn2+ and low pH inhibit Cl- efflux by separate and distinct mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrate and chloride ions have different permeation pathways in skeletal muscle fibers of Rana pipiens.

The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined. Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K2SO4 solutions. External solutions were first changed to ones in which either Rb+ or Cs+ replaced K+ and then to solutions containing either NO3- or Cl- to replace SO4(2-). The hyperpolarizations produced by Cl- depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO3- were independent of external pH over a pH range from 5.5 to 9.0. In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K2SO4 plus Na2SO4. KCl or KNO3 were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K+ was replaced by TEA+. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl- or NO3- depending on the final equilibrating solution. Cl- currents during voltage pulses were both external pH and time dependent. By contrast, NO3- currents were independent of both external pH and time. The voltage dependence of NO3- currents could be fit by constant field equations with a PNO3 of 3.7.10(-6) cm/sec. The voltage dependence of the initial or "instantaneous" Cl- currents at pH 7.5 and 9.0 could also be fit by constant field equations with PCl of 5.8 x 10(-6) and 7.9 x 10(-6) cm/sec, respectively. At pH 5.0, no measurable "instantaneous" Cl- currents were found. From these results we conclude that NO3- does not pass through the pH, time-dependent Cl- channels but rather passes through a distinct set of channels. Furthermore, Cl- ions do not appear to pass through the channels which allow NO3- through. Consequently, the measured ratio of PCl/PNO3 based on membrane potential changes to ionic changes made on intact skeletal muscle fibres is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles.

Efflux of 42K+ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K+ concentrations [( K+]0) between 75 and 300 mM and NaCl concentrations of 60, 120, or 240 mM. For several combinations of KCl and NaCl, steady-state internal potentials (Vi) were the same for different [K+]0. For the range of Vi examined, K+ efflux occurs principally through the K+ inward rectifier channels. When external K+ is removed Vi remains constant for 2 to 3 hr because of the high membrane conductance to Cl-, but K+ efflux drops by about one order of magnitude. External Ba2+ in the presence or absence of external K+ produces an inhibition of K+ efflux described by a relation of the form u = (u1/(1 + C [Ba2+]0] + u2, where u is the uninhibited fraction of K+ efflux; u1, u2 and C are constants; and u1 + u2 = 1. C depends both on [K+]0 and Vi. When [K+]0 greater than or equal to 75 mM, increasing [K+]0 at constant Vi reduces Ba2+ sensitivity. For constant Vi greater than or equal to -30 mV, Ba2+ sensitivity is less when [K+]0 = 0 than when [K+]0 greater than or equal to 75 mM. When [K+]0 = 0, Ba2+ sensitivity decreases as Vi is made more positive. The dependence of the Ba2+ sensitivity on Vi at constant [K+]0 is greater when [K+]0 = 0 than when [K+]0 greater than or equal to 75 mM. Both the activation of K+ efflux by external K+ and the Ba2+ inhibition of K+ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K+, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba2+ occupies either site, K+ efflux is further reduced. The reduction of Ba2+-sensitivity by increasing [K+]0 at high [K+]0 is attributable to the displacement of Ba2+ from the control sites by K+. The increased Ba2+ sensitivity produced by going from [K+]0 = 0 to [K+] greater than or equal to 75 mM when Vi greater than or equal to -30 mV is attributable to states in which Ba2+ occupies one site and K+ the other when [K+]0 not equal to 0.(ABSTRACT TRUNCATED AT 400 WORDS)

Zinc inhibition of potassium efflux in depolarized frog muscle and its modification by external hydrogen ions and diethylpyrocarbonate treatment.

Efflux of 42K+ was measured in frog sartorius muscles equilibrated in hyperosmotic depolarizing solutions. At the internal potentials obtained, K+ passes mainly through the inward rectifier potassium channels. Inhibition of K+ efflux by external Zn2+ (0.25 to 15 mM) differs in three significant ways from inhibition by Ba2+. (1) The dose-response relation does not correspond to action at a single site. (2) The Zn2+-sensitivity of K+ efflux does not depend on [K+]0 at constant internal potential. (3) Zn2+ inhibition is reduced by hydrogen ions, while Ba2+ inhibition is unaffected. Further, the Ba2+-sensitivity of K+ efflux is not altered by a half-inhibiting Zn2+ concentration, suggesting that the two ions do not interact at a common site. The histidine-modifying reagent diethylpyrocarbonate (DEPC) reduces Zn2+ inhibition. After DEPC treatment Zn2+ inhibition is further reduced by low pH. DEPC has little effect on Ba2+ inhibition. Zn2+ inhibition is not altered by treatment with the sulfhydryl reagents 5,5'-dithio-bis(2-nitrobenzoic acid) or dithiothreitol. The results can be described by either of two models in which two sites can bind Zn2+ and one or both of the sites may also bind H+. When both sites bind Zn2+, K+ efflux is inhibited, and a third site may then bind H+. The effects of DEPC can be accounted for by a decrease in H+ affinity of the first two sites by a factor of 50, and a decrease in Zn2+ affinity of these sites and of the H+ affinity of the third site by about one order of magnitude.

Depolarization-contraction coupling in short frog muscle fibers. A voltage clamp study.

Short muscle fibers (1.5 mm) were dissected from hindlimb muscles of frogs and voltage clamped with two microelectrodes to study phenomena related to depolarization-contraction coupling. Isometric myograms obtained in response to depolarizing pulses of durations between 10 and 500 ms and amplitudes up to 140 mV had the following properties. For suprathreshold pulses of fixed duration (in the range of 20-100 ms), the peak tension achieved, the time to peak tension, and contraction duration increased as the internal potential was made progressively more positive. Peak tension eventually saturates with increasing internal potentials. For pulse durations of greater than or equal to 50 ms, the rate of tension development becomes constant for increasing internal potentials when peak tensions become greater than one-third of the maximum tension possible. Both threshold and maximum steepness of the relation between internal potential and peak tension depend on pulse duration. The relation between the tension-time integral and the stimulus amplitude-duration product was examined. The utility of this relation for excitation-contraction studies is based on the observation that once a depolarizing pulse configuration has elicited maximum tension, further increases in either stimulus duration or amplitude only prolong the contractile response, while the major portion of the relaxation phase after the end of a pulse is exponential, with a time constant that is not significantly affected by either the amplitude or the duration of the pulse. Hence, the area under the tension-response curve provides a measure of the availability to troponin of the calcium released from the sarcoplasmic reticulum in response to membrane depolarization. The results from this work complement those obtained in experiments in which intramembrane charge movements related to contractile activation were studied and those in which intracellular Ca++ transients were measured.

Denervated frog skeletal muscle. Some electrical and mechanical properties.

The effects of denervation on several mechanical and electrical parameters of frog sartorius muscle have been investigated. In denervated muscles, there is no change in the resting potential and a relatively small change in the action potential. The first alteration in the action potential is a reduction of about 30% in the maximum rate of repolarization in muscles that have been denervated for 40 days or longer. Later, the overshoot and maximum rate of depolarization also decline. No tetrodotoxin resistant action potentials could be detected. Fibrillatory potentials were observed infrequently and in most cases in depolarized fibers. Twitch tension is significantly reduced by denervation while the tetanus tension is practically unaffected by denervation. The experiments suggest that the decline in twitch tension produced by denervation reflect a defect in some step of the excitation contraction coupling sequence. On the other hand, post-tetanic potentiation of the twitch is much larger in denervated than in control muscles. This potentiation in denervated muscles is paralleled by an increased action potential duration which returns to its pretetanic duration with a time course indistinguishable from that of the twitch potentiation.

Caffeine contractures in denervated frog muscle.

Caffeine contracture tension, effect of caffeine on the resting membrane potential, and caffeine influx in normal and denervated frog sartorius muscle have been investigated. Peak caffeine contracture tension is increased after denervation at all caffeine concentrations. The percentage increases in tension are highest for lower caffeine concentrations. The caffeine concentration required for half maximum tension is decreased from about 3.6 mM in control muscles to 2.6 mM in denervated muscles. Caffeine at 3.5 mM produces a depolarization of about 6 mV in control muscles and 16mV in denervated muscles. The large contracture tensions observed in denervated muscles are not due to the greater depolarization produced by the drug in denervated muscles since innervated muscles depolarized to the same level by external K+ do not enhance caffeine contracture tension. Both control and denervated muscles are highly permeable to caffeine. The increases in sarcoplasmic reticulum development ( Moscatello et al. 1965) and calcium content ( Picken and Kirby 1976) promoted by denervation may explain the larger tension elicited by caffeine in denervated muscles.

Inhibition of tumor cell attachment to concanavalin A-coated surfaces as an assay for teratogenic agents: approaches to validation.

Four environmental agents have been tested for activity in a recently developed in vitro teratogen assay system. All four agents inhibited attachment. The agents were 40-fold concentrated drinking water (ID50 = 0.45 ml/ml), whole cigarette smoke condensate (ID50 = 85 micrograms/ml), kerosene soot (ID50 = 90 micrograms/ml), and commercial formulations of the pesticide carbaryl (ID50 approximately 150 micrograms/ml). On the basis of these examples appropriate criteria for the validation of in vitro teratogen assay systems are discussed. It is concluded that criteria are critically dependent on the specific applications of the assay system. For example, the false-positive rate must be minimized to make a wide-ranging screen of water samples useful. On the other hand, an investigation of impurities in commercial compounds requires low false-negative rates. In every case a quantitative measure of the potential teratogenic potency, in vivo, is desirable.

The dual effect of rubidium ions on potassium efflux in depolarized frog skeletal muscle.

The effects of external Rb+ on the efflux of 42K+ from whole frog sartorius muscles loaded with 305 mM K+ and 120 mM Cl- were studied. K+ efflux is activated by [Rb+]o less than about 40 mM according to a sigmoid relation similar to that for activation by [K+]o. At [Rb+]o greater than 40 mM, K+ efflux declines, although at [Rb+]o = 300 mM it is still greater than at [Rb+]o = 0 mM. For low concentrations, the increment in K+ efflux over that in K+-and Rb+-free solution, delta K, is described by the relation delta k = a[X+]on, for both K+ and Rb+. The value of a is larger for Rb+ than for K+, while the values of n are similar; the activation produced by a given [Rb+]o is larger than that by an equal [K+]o for concentrations less than about 40 mM. Adding a small amount of Rb+ to a K+-containing solution has effects on K+ efflux which depend on [K+]o. At low [K+]o, adding Rb+ increases K+ efflux, the effect being greatest near [K+]o = 30 mM and declining at higher [K+]o; at [K+]o above 40 mM, addition of Rb+ decreases K+ efflux. At [K+]o above 75 mM, where K+ efflux is largely activated, Rb+ reduces K+ efflux by a factor b, described by the relation b = 1/(1+c[Rb+]o). Activation is discussed in terms of binding to at least two sites in the membrane, and the reduction in K+ efflux by Rb+ at high [K+]o in terms of association with an additional inhibitory site.

Unidirectional flux ratio for potassium ions in depolarized frog skeletal muscle.

Small bundles of frog skeletal muscle fibers were loaded with 305 mM K+ and 120 mM Cl-, and 42K+ tracer efflux and influx were measured as a function of external K+ concentration ([K+]o) at a resting potential of -2 mV. As [K+]o was lowered from 305 mM, efflux decreased along a markedly sigmoidal curve, reaching a constant nonzero value at low [K+]o. Influx varied linearly with [K+]o at low [K+]o and more steeply at higher [K+]o. The ratio of influx to efflux was described by the equation: influx/efflux = exp[-n(V - VK)F/RT] with n = 2 at high [K+]o, but the ratio approached this equation with n = 1 at low [K+]o. Efflux did not depend on [K+]o when the membrane potential was raised to +36 mV, whereas at low [K+]o decreasing the membrane potential to -19 mV further activated the efflux. The results are discussed in terms of an inwardly rectifying potassium channel with two or more activating sites within the membrane that bind K+ and are accessible from the external solution.

Membrane charge movement in contracting and non-contracting skeletal muscle fibres.

1. The single gap voltage clamp technique (Kovács & Schneider, 1978) was used to monitor membrane charge movement in tendon-terminated short segments of cut frog skeletal muscle fibres.2. Experiments were performed both on fibres able to contract and on fibres in which contraction was eliminated by exposing the open end to a solution containing 20 mm-EGTA. In both cases ionic conductances were minimized by using a predominantly caesium glutamate solution at the open end and a predominantly tetraethyl-ammonium sulphate solution with tetrodotoxin at the closed end.3. Modifications of previously used charge movement analysis procedures included synthesis of a ;mean linear' ON and OFF capacitative transient from the OFFs of several different hyperpolarizing pulses and use of only the first 35 msec of the ;mean linear' transient so that base lines could be fitted to unaltered latter parts of ON and OFF currents for depolarizing pulses.4. Simultaneous two-micro-electrode and gap current recording from gap-clamped fibres with blocked contraction established the validity of gap-recorded charge movement currents.5. For pulses to below about 0 mV in non-contracting fibres the charges Q(ON) and Q(OFF) moved by the non-linear transient currents at pulse ON and OFF were approximately equal. For pulses to between about 0 and +50 mV Q(OFF) exceeded Q(ON), with the charge inequality increasing with both pulse amplitude and pulse duration.6. Use of 20 mm-cobalt in the solution at the closed end eliminated the ON:OFF charge inequality for large depolarizations by decreasing Q(OFF).7. The charge inequality and cobalt effect indicate that, in the absence of cobalt, ionic conductance was being slowly activated during depolarizations to between 0 and +50 mV and that inward calcium current tails were contributing to the measured Q(OFF) values. The small and slowly developing ionic current during large depolarizations was probably removed with the straight sloping base line so that Q(ON) was minimally affected by conductance activation.8. Average Q vs. V results for pulses to at most 0 mV in eighteen non-contracting fibres were well fitted by the two-state Boltzmann model where Q = Q(max)/[1+exp-(V - V)/k] with Q(max) = 26.7+/-0.6 nC/muF, k = 16.7+/-0.6 mV and V = -32.9+/-1.0 mV (least-squares values+/-s.d. obtained from fit).9. In contracting fibres the only apparent artifact produced by contraction in the I(Q) records for pulses to at most 0 mV was a ;bowing' of the OFF base lines for the larger pulses. The ON records appeared to be unaffected by contraction artifacts.10. The average Q vs. V relationship for pulses to at most 0 mV in contracting fibres was virtually identical to the one obtained from fibres in which contraction was blocked.11. The ON portions of I(Q) records for pulses to between about -50 and -25 mV exhibited prolonged tails, plateaux or secondary rising phases whereas the OFF portions decayed smoothly. I(Q) time courses were not noticeably different with or without blockage of contraction by internal EGTA.

Membrane charge moved at contraction thresholds in skeletal muscle fibres.

1. The current I(Q) due to membrane charge movement and the threshold pulse duration t(th) required to produce microscopically just-detectable contraction were determined for pulses to a variety of membrane potentials in tendon-terminated short segments of cut frog skeletal muscle fibres voltage-clamped using a single gap technique.2. The time course Q(t) of membrane charge movement was calculated as the running integral of I(Q). The threshold charge Q(th) moved by pulses which produced just-detectable contraction was estimated as Q(t(th)).3. Q(th) was constant for pulses to potentials ranging from about -45 mV, the rheobase potential for contraction, to about -15 mV, where t(th) was about 9 msec. The mean Q(th) from fourteen fibres was 11.5 nC/muF, when the holding potential was about -100 mV.4. Prepulses of 50 msec which were themselves sub-rheobase for producing contraction decreased the t(th) for an immediately following test pulse. The total threshold charge moved during the prepulse and during t(th) of the test pulse was equal to Q(th) for the test pulse without prepulse.5. Items 3 and 4 above indicate that t(th) is determined by the time required to move a set amount of intramembrane charge, independent of the kinetics of the charge movement.6. Steady partial fibre depolarization to between -70 and -55 mV increased t(th) at all membrane potentials and elevated the rheobase potential for contraction. Slight further steady depolarization totally eliminated contraction.7. Steady partial depolarization decreased the total ON charge movement Q(ON) by about the same factor for pulses to all potentials tested.8. Q(th) for partially depolarized but still-contracting fibres remained approximately independent of membrane potential from rheobase to about 0 mV but was slightly less than Q(th) for the same fibres when fully polarized.9. Steady partial depolarizations which reduced the mean (+/-s.d.) ON charge movement Q(ON) to 60 +/- 8% of its value under full polarization reduced Q(th) to 86 +/- 11% of its full polarization value (n = 10). These steady partial depolarizations produced no change in the linear capacitance measured with hyperpolarizing pulses.10. Contraction was completely abolished by steady partial depolarizations which reduced Q(ON) to 41% of its value under full polarization (mean of three runs). The maximum value of Q(ON) was then 77% of the Q(th) value for the same fibres under full polarization.11. A prolonged tail, a shoulder, a second rising phase or an early relatively high flat segment were successively evident in the I(Q) records as the depolarizing pulse was successively increased to and beyond the rheobase potential for contraction. It was found that t(th) either coincided with or occurred slightly later than the start of such tails, shoulders or second rising phases.12. When test pulse I(Q) records with and without immediately preceding sub-rheobase prepulses were shifted in time so that their t(th) times coincided, the record with prepulse coincided with the later part of I(Q) without prepulse. This indicates that sub-rheobase prepulses moved the initial portion of the I(Q) that occurs during the test pulse alone, whereas they did not alter the latter portion of the test pulse I(Q).13. A model was developed which accounts for charge movement's voltage dependence and kinetics and for the relationship between charge movement and just-detectable contraction in both the fully polarized and partially depolarized states.14. The model proposes that Q be composed of two components. Component A is due to the voltage and time-dependent movement of charges between two sites located within the membrane and separated by a single energy barrier. Component B is instantaneously proportional to an integer power n of the fraction of component A charges which have crossed the barrier.15. The I(Q) time courses were best approximated using n = 3, with which both the relatively early and late portions of the experimental I(Q) time courses could be reproduced. The best theoretical records obtained with n = 3 still passed below the shoulders, second rising phases and later parts of the early constant phases in the various experimental I(Q) records. Theoretical records did fit accurately the I(Q) time courses observed under steady partial fibre depolarization. The relatively small current not reproduced by the model may be an electrical accompaniment of the activation of calcium release or the elevation of internal free calcium levels in the space between the transverse tubules (T-tubules) and the sarcoplasmic reticulum.

Density and apparent location of the sodium pump in frog sartorius muscle.

The binding of the cardiosteroid 3H-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release. The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding (Bmax) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant (K) of 2.2 X 10(-7)M. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one. Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (tau approximately equal to 25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (tau approximately equal to 15 hr). Increasing [K+]O from 2.5 to 10 mM slows the time course of binding without any significant change in binding capacity of the muscle fibers. Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979) Biochim. Biophys. Acta 552:247] on the distribution of (Na+ + K+)- ATPase among different cell membrane fractions from frog skeletal muscle.

Theophylline action on sodium fluxes in frog striated muscle.

The action of theophylline on both sodium efflux and influx was measured using freshly isolated frog sartorius muscles. In normal Ringer's fluid, 2 mM theophylline increased sodium efflux by 35% whereas it decreased sodium influx by about 10%. The percent increase in sodium efflux produced by 2 mM theophylline was not significantly altered in sodium-free, lithium-containing solutions. Strophanthidin prevented the stimulation of sodium efflux by 2 mM theophylline in both normal, sodium-containing Ringer's fluid and sodium-free, lithium-containing solutions. Hence, the major effect of theophylline seems to be stimulation of active sodium transport and the enhanced rate of sodium exit induced by theophylline does not seem to require the presence of external sodium. An interesting and unexplained findings is that 2 mM theophylline, which does not produce a maximal stimulation of sodium efflux, prevents the increased sodium efflux induced by saturating doses of epinephrine.

Density and distribution of tetrodotoxin receptors in normal and detubulated frog sartorius muscle.

Tetrodotoxin (TTX) binding was measured in muscles which were either in normal condition or which had been "detubulated" by glycerol-induced osmotic shock. In both cases the binding of TTX was found to saturate at high TTX concentrations. Maximum binding in normal fibers was 35 pmol/g wet weight, and that figure was reduced to 16 pmol/g after glycerol treatment. The dissociation constant for binding to the surface membrane was 3 nM, which is the range of values obtained by electrophysiological measurements of the effect of TTX on the maximum rate of rise of the action potential. The results suggest that the dissociation constant in the transverse tubules may be higher than that in the surface. Control experiments indicate that the effects of glycerol treatment are limited to the accessibility of the receptors to the toxin and result in no alteration of the affinity of the binding site. TTX receptors in the transverse tubules may be recovered after glycerol treatment by homogenization of the fibers. The measurements suggest that the density of sodium channels in surface membrane is about 175/muM2 and that in the transverse tubular membrane is 41-52/mum2.

Fluorescence intensity changes associated with contractile activation in frog muscle stained with Nile Blue A.

1. Extrinsic fluorescence intensity changes were studied in frog semitendinosus muscles stained with Nile Blue A in response to electrical stimulation. Muscles were stretched and put into hypertonic solutions to prevent movement. The muscles were illuminated at 90 degrees to their long axis with a narrow beam of light at a central wave-length of 6250 . Fluorescence emission was measured at 90 degrees to the exciting light using a filter which absorbed light of wave-lengths shorter than 6400 . 2. In response to a single stimulus the fluorescence intensity increases briefly. The fluorescence response is propagated at a constant velocity of about 1.5 m/sec. The average ratio of the maximum fluorescence intensity change to the resting fluorescence is 4.5 times 10-3 for supramaximal shocks. The fluorescence intensity change starts early in the falling phase of the action potential. 3. The fluorescence intensity change increases when nitrate replaces chloride and decreases when D2O replaces H2O. The rates of rise and fall of the fluorescence change was unaffected by nitrate replacement of chloride but are slowed where D2O replaces H2O. The rates of rise and fall of the fluorescence change increase with increasing temperature for all solutions used. The peak fluorescence intensity change, however, goes through a maximum at about 17 degrees C for aqueous chloride and nitrate solutions in the range of 10-25 degrees C. With D2O solutions, the peak fluorescence intensity increases monotonically in this range of temperatures. 4. The fluorescence intensity change in response to trains of action potentials are not additive. 5. Depolarization of muscles treated with tetrodotoxin using triangular-shaped fluid electrodes produces an increase in fluorescence at about the same threshold values required to elicit tension in preparations that are not fully stretched. The fluorescence intensity change precedes in time tension development. Near threshold depolarizations, the delay in onset of the fluorescence response can be 80 msec or longer. Byond threshold, delays become shorter and peak responses larger. During maintained depolarization, after the peak response, fluorescence declines to a plateau value. 6. The results suggest that the fluorescence intensity changes are associated with excitation-contraction coupling, possibly with changes in the transmembrane potential of the sarcoplasmic reticulum.