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Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ∼ 11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (C(cb)) and filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa(-1), close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa(-1)). C(cb) calculated using these estimates is 0.37 ± 0.12 nm pN(-1), a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN(-1), obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to C(cb) from the myosin tail that joins the head to the thick filament is excluded. The low C(cb) value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre-microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4 °C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa(-1) (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model.

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

Rapid attachment to actin of the detached motor domain of myosin dimers with one motor domain already attached has been hypothesized to explain the stretch-induced changes in X-ray interference and stiffness of active muscle. Here, using half-sarcomere mechanics in single frog muscle fibres (2.15 microm sarcomere length and 4 degrees C), we show that: (1) an increase in stiffness of the half-sarcomere under stretch is specific to isometric contraction and does not occur in rigor, indicating that the mechanism of stiffness increase is an increase in the number of attached motors; (2) 2 ms after 100 micros stretches (amplitude 2-8 nm per half-sarcomere) imposed during an isometric tetanus, the stiffness of the array of myosin motors in each half-sarcomere (e(m)) increases above the isometric value (e(m0)); (3) e(m) has a sigmoidal dependence on the distortion of the motor domains (Delta z) attached in isometric contraction, with a maximum approximately 2 e(m0) for a distortion of approximately 6 nm; e(m) is influenced by detachment of motors at z > 6 nm; (4) at the end of the 100 micros stretch the relation between e(m)/e(m0) and Delta z lies slightly but not significantly above that at 2 ms. These results support the idea that stretch-induced sliding of the actin filament distorts the actin-attached motor domain of the myosin dimers away from the centre of the sarcomere, providing the steric conditions for rapid attachment of the second motor domain. The rate of new motor attachment must be as high as 7.5 x 10(4) s(1) and explains the rapid and efficient increase of the resistance of active muscle to stretch.

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, Pc) and the sarcomere length needed to reach Pc (critical length, Lc) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that Pc was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, Pc was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 +/- 0.17 nm per half-sarcomere (nm hs(-1)) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, Lc reduced to 8.19 +/- 0.039 nm hs(-1). Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.

The aims of this study were to investigate the effects of solution tonicity on muscle properties, and to verify their consistence with the lever arm theory of force generation. Experiments were made in single muscle fibres and in fibre bundles from the frog, using both fast stretches and time-resolved X-ray diffraction, in isotonic Ringer solution (1T), hypertonic (1.4T) and hypotonic (0.8T) solutions. Fast stretches (0.4-0.6 ms duration and 16-25 nm per half-sarcomere (nm hs(-1)) amplitude) were applied at various tensions during the force development in isometric tetani. Force increased during the stretch up to a peak (critical tension, Pc) at which it started to fall, in spite of continued stretching. In all solutions, Pc was proportional to the initial isometric tension developed. For a given isometric tension, Pc increased with solution tonicity and occurred at a precise sarcomere elongation (critical length, Lc) which also increased with tonicity. M3 meridional layer line intensity (I M3) was measured during the application of sinusoidal length oscillations (1 kHz frequency, and about 2% fibre length amplitude) at tetanus plateau. I M3 changed during the length oscillations in a sinusoidal manner in phase opposition to length changes, but a double peak distortion occurred at the peak of the release phase. The presence of the distortion, which decreased with tonicity, allowed calculation of the mean position of the myosin head (S1) during the oscillation cycle. In agreement with the lever arm theory, both X-ray diffraction and mechanical data show that solution tonicity affects S1 mean position and consequently crossbridge individual extension and force, with no effect on crossbridge number. The force needed to break the single crossbridge was insensitive to solution tonicity suggesting a non-ionic nature of the actomyosin bond.

Very fast ramp stretches at 9.5-33 sarcomere lengths s(-1) (l0 s(-1)) stretching speed, 16-25 nm per half-sarcomere (nm hs(-1)) amplitude were applied to activated intact frog muscle fibres at tetanus plateau, during the tetanus rise, during the isometric phase of relaxation and during isotonic shortening. Stretches produced an almost linear tension increase above the isometric level up to a peak, and fell to a lower value in spite of continued stretching, indicating that the fibre became suddenly very compliant. This suggests that peak tension (critical tension, P(c)) represents the tension at which crossbridges are forcibly detached by the stretch. The ratio of P(c) to the isometric tension at tetanus plateau (P0) was 2.37 +/- 0.12 (S.E.M.). This ratio did not change significantly at lower tension (P) during the tetanus rise but decreased with time during the relaxation and increased with speed during isotonic shortening. At tetanus plateau P(c) occurred when sarcomere elongation attained a critical length (L(c)) of 10.98 +/- 0.13 nm hs(-1), independently of the stretching speed. L(c) remained constant during the tetanus rise but decreased on the relaxation and increased during isotonic shortening. Length-clamp experiments on the relaxation showed that the lower values of P(c)/P ratio and L(c), were both due to the slow sarcomere stretching occurring during this phase. Our data show that P(c) can be used as a measure of crossbridge number, while L(c) is a measure of crossbridge mean extension. Accordingly, for a given tension, crossbridges on the isometric relaxation are fewer than during the rise, develop a greater individual force and have a greater mean extension, while during isotonic shortening crossbridges are in a greater number but develop a smaller individual force and have a smaller extension.

Spontaneous membrane voltage oscillations were found in 27 of 130 isolated frog saccular hair cells. Voltage oscillations had a mean peak-to-peak amplitude of 23 mV and a mean oscillatory frequency of 4.6 Hz. When compared with non-oscillatory cells, oscillatory cells had significantly greater hyperpolarization-activated and lower depolarization-activated current densities. Two components, the hyperpolarization-activated cation current, I(h), and the K(+)-selective inward-rectifier current, I(K1), contributed to the hyperpolarization-activated current, as assessed by the use of the I(K1)-selective inhibitor Ba(2+) and the I(h)-selective inhibitor ZD-7288. Five depolarization-activated currents were present in these cells (transient I(BK), sustained I(BK), I(DRK), I(A), and I(Ca)), and all were found to have significantly lower densities in oscillatory cells than in non-oscillatory cells (revealed by using TEA to block I(BK), 4-AP to block I(DRK), and prepulses at different voltages to isolate I(A)). Bath application of either Ba(2+) or ZD-7288 suppressed spontaneous voltage oscillations, indicating that I(h) and I(K1) are required for generating this activity. On the contrary, TEA or Cd(2+) did not inhibit this activity, suggesting that I(BK) and I(Ca) do not contribute. A mathematical model has been developed to test the interpretation derived from the pharmacological and biophysical data. This model indicates that spontaneous voltage oscillations can be generated when the electrophysiological features of oscillatory cells are used. The oscillatory behaviour is principally driven by the activity of I(K1) and I(h), with I(A) playing a modulatory role. In addition, the model indicates that the high densities of depolarization-activated currents expressed by non-oscillatory cells help to stabilize the resting membrane potential, thus preventing the spontaneous oscillations.

The dihydropyridine receptors (DHPRs)/L-type Ca2+ channels of skeletal muscle are coupled with ryanodine receptors/Ca2+ release channels (RyRs/CRCs) located in the sarcoplasmic reticulum (SR). The DHPR is the voltage sensor for excitation-contraction (EC) coupling and the charge movement component q gamma has been implicated as the signal linking DHPR voltage sensing to Ca2+ release from the coupled RyR. Recently, a new charge component, qh, has been described and related to L-type Ca2+ channel gating. Evidence has also been provided that the coupled RyR/CRC can modulate DHPR functions via a retrograde signal. Our aim was to investigate whether the newly described qh is also involved in the reciprocal interaction or cross-talk between DHPR/L-type Ca2+ channel and RyR/CRC. To this end we interfered with DHPR/L-type Ca2+ channel function using nifedipine and 1-alkanols (heptanol and octanol), and with RyR/CRC function using ryanodine and ruthenium red (RR). Intramembrane charge movement (ICM) and L-type Ca2+ current (ICa) were measured in single cut fibres of the frog using the double-Vaseline-gap technique. Our records showed that nifedipine reduced the amount of q gamma and qh moved by approximately 90% and approximately 55%, respectively, whereas 1-alkanols completely abolished them. Ryanodine and RR shifted the transition voltages of q gamma and qh and of the maximal conductance of ICa by approximately 4-9 mV towards positive potentials. All these interventions spared q beta. These results support the hypothesis that only q gamma; and qh arise from the movement of charged particles within the DHPR/L-type Ca2+ channel and that these charge components together with ICa are affected by a retrograde signal from RyR/CRC.

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the beta2-adrenergic stimulation of the L-type Ca2+ current (ICa,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 microM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of ICa,L were used to determine the gradient of local vs. distant cAMP concentration (alpha). IBMX (100 microM), a non-selective PDE inhibitor, reduced alpha from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 microM) or PDE4 inhibition by Ro 20-1724 (3 microM) reduced alpha by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 microM), a non-selective phosphatase inhibitor, or H89 (1 microM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of ICa,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon beta2-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of beta2-adrenergic receptors with nearby Ca2+ channels via local elevations of cAMP.

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanized intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (approximately 70 %, from 2 to 17 degrees C); (2) increases the rate of quick force recovery following small length steps (range between -3 and 2 nm (half-sarcomere)-1) with a Q10 (between 2 and 12 degrees C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)-1). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.

Myosin II is the motor protein that produces force and shortening in muscle by ATP-driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin II's working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 micros force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head.

1. A contracting muscle resists stretching with a force greater than the force it can exert at a constant length, T(o). If the muscle is kept active at the stretched length, the excess tension disappears, at first rapidly and then more slowly (stress relaxation). The present study is concerned with the first, fast tension decay. In particular, it is still debated if and to what extent the fast tension decay after a ramp stretch involves a conservation of the elastic energy stored during stretching into cross-bridge states of higher chemical energy. 2. Single muscle fibres of Rana temporaria and Rana esculenta were subjected to a short ramp stretch (approximately 15 nm per half-sarcomere at either 1.4 or 0.04 sarcomere lengths s(-1)) on the plateau of the force-length relation at temperatures of 4 and 14 degrees C. Immediately after the end of the stretch, or after discrete time intervals of fixed-end contraction and stress relaxation at the stretched length (Delta t(isom) = 0.5-300 ms), the fibre was released against a force ~T(o). Fibre and sarcomere stiffness during the elastic recoil to T(o) (phase 1) and the subsequent transient shortening against T(o) (phase 2), which is expression of the work enhancement by stretch, were measured after different Delta t(isom) and compared with the corresponding fast tension decay during Delta t(isom). 3. The amplitude of fast tension decay is large after the fast stretch, and small or nil after the slow stretch. Two exponential terms are necessary to fit the fast tension decay after the fast stretch at 4 degrees C, whereas one is sufficient in the other cases. The rate constant of the dominant exponential term (0.1-0.2 ms(-1) at 4 degrees C) increases with temperature with a temperature coefficient (Q(10)) of approximately 3. 4. After fast stretch, the fast tension decay during Delta t(isom) is accompanied in both species and at both temperatures by a corresponding increase in the amplitude of phase 2 shortening against T(o) after Delta t(isom): a maximum of approximately 5 nm per half-sarcomere is attained when the fast tension decay is almost complete, i.e. 30 ms after the stretch at 4 degrees C and 10 ms after the stretch at 14 degrees C. After slow stretch, when fast tension decay is small or nil, the increase in phase 2 shortening is negligible. 5. The increase in phase 2 work during fast tension decay (Delta W(out)) is a constant fraction of the elastic energy simultaneously set free by the recoil of the undamped elastic elements. 6. Delta W(out) is accompanied by a decrease in stiffness, indicating that it is not due to a greater number of cross-bridges. 7. It is concluded that, during the fast tension decay following a fast ramp stretch, a transfer of energy occurs from the undamped elastic elements to damped elements within the sarcomeres by a temperature-dependent mechanism with a dominant rate constant consistent with the theory proposed by A. F. Huxley and R. M. Simmons in 1971.

1. The regulation of L-type Ca(2+) current (I(Ca)) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 microM to 1 mM) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 microM) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques. 2. SNP and SNAP depressed the isoprenaline (ISO, 10-100 nM)- or forskolin (FSK, 1 microM)-mediated stimulation of I(Ca) via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nM) response was observed at 1 mM SNP. 3. When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of I(Ca) to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mM SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mM SNP). 4. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mM) reversed the local effect of SNAP (3 microM) on FSK-stimulated I(Ca) when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. 5. A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 microM), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 microM) or SNAP (10 microM) on I(Ca) but had no effect on the distant response. 6. When EHNA was applied on the distant side, with SNP (1 mM) and ISO (100 nM) applied locally, the distant effect of SNP was fully reversed. 7. Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca(2+) channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca(2+) channels and PDE2.

The cAMP fluorescent probe FlCRhR was used to monitor changes in intracellular cAMP concentration ([cAMP]i) in isolated frog ventricular myocytes. The probe was introduced into the cell through a patch pipette which allowed simultaneous recording of the whole-cell L-type Ca2+ current (ICa). Ratiometric imaging was used to monitor [cAMP]i changes in response to the beta-adrenergic agonist isoprenaline (ISO) or to the direct adenylyl cyclase activator forskolin (FSK). FlCRhR fluorescence was distributed in the cytosol in a striated pattern, with high fluorescence in the I-bands and low fluorescence in the A-bands. This pattern of distribution was mimicked by fluorescein dextran, another high molecular weight fluorescent molecule, and was therefore likely to be due to anisotropic diffusion of the probe in the cytosol due to the hindrance generated by sarcomeric proteins in the A-bands. Introduction of FlCRhR into the cell induced a small approximately 70% stimulatory effect on basal ICa, attenuating about 2-fold a subsequent response of ICa to 1-10 microM ISO (from 400 to 200%). Brief (10 s) application of a saturating concentration of ISO (1-20 microM) to the cell induced a transient increase in both ICa and [cAMP]i. However, the [cAMP]i transient was approximately 2-fold shorter in duration than the ICa transient, i.e. ICa was still strongly enhanced when [cAMP]i had already returned to control level. This indicates that hydrolysis of cAMP by phosphodiesterases is not a rate limiting step in the recovery of ICa from ISO stimulation. When the application of ISO was maintained, ICa and [cAMP]i responses followed a similar time course, with a half-maximal response at approximately 60 s. This suggests that activation of Ca2+ channels by cAMP-dependent protein kinase occurs on a much faster time scale than the rise in [cAMP]i. When the cells were exposed to FSK (13 microM), both responses of ICa and [cAMP]i were approximately 2-fold slower than with ISO. This demonstrates that the slower response of ICa to FSK is due to a slower rise in [cAMP]i rather than to some inhibitory effect of FSK on ICa or to a direct or priming effect of the stimulatory G protein Gs on Ca2+ channels. Simultaneous measurements of [cAMP]i and ICa changes in intact cardiac myocytes opens the way to dissect the temporal sequence of events in the cAMP cascade mediating the response of the heart to a large number of hormones and inotropic agents.

1. We report opposite inotropic effects of NO donors in frog cardiac fibres. The negative effect, elicited by either 3-morpholino-sydnonimine (SIN-1) or S-nitroso-N-acetyl-penicillamine (SNAP), involved cyclic GMP (cGMP) production. However, SIN-1, unlike SNAP, could elicit a positive effect, in a superoxide dismutase (SOD)-sensitive manner. SIN-1, unlike SNAP, can release both NO and superoxide anion, the precursors of peroxynitrite (OONO-). The role of these messengers was examined. 2. Catalase did not reduce the positive inotropic effect of SIN-1. Thus, a conversion of superoxide anion into hydrogen peroxide was not involved in this effect. In addition, catalase did not modify the negative effects of SIN-1 plus SOD, or SNAP plus SOD. 3. LY 83583, a superoxide anion generator, elicited a positive inotropic effect, like SIN-1. The effect of LY 83583 was additive to the negative effects of SIN-1 or SNAP, and to the positive effect of SIN-1. Thus, superoxide anion generation, per se, did not account for the positive effect of SIN-1. 4. Authentic peroxynitrite (OONO-), but not mock-OONO- (negative control plus decomposed OONO-), exerted a dramatic positive inotropic effect in cardiac fibres. The effect of OONO- was larger in atrial fibres, as compared with ventricular fibres. 5. The positive effect of OONO- was not additive with that of SIN-1, suggesting a common mechanism of action. In contrast, the effects of either OONO- or SIN-1 were additive with the negative inotropic effect of SNAP. Furthermore, the effect of OONO-, like that of SIN-1, was not antagonized by 1H-[1,2,4]xidiazolo[4, 3-a]quinoxaline-1-one (ODQ; 10 microM), the guanylyl cyclase inhibitor. 6. The positive inotropic effects of SIN-1 and OONO- were not modified by hydroxyl radical scavengers, such as dimethyl-thio-urea (DMTU; 10 mM). 7. The positive inotropic effect of SIN-1 (100 microM) was abolished in sodium-free solutions, a treatment that eliminates the activity of the sodium-calcium exchanger. In contrast, the effect of SIN-1 was unchanged by a potassium channel inhibitor (tetraethyl-ammonium, 20 mM), or a sodium-potassium pump inhibitor (ouabain 10 microM). 8. We conclude that OONO- is a positive inotropic agent in frog cardiac fibres. The generation of OONO- accounts for the positive inotropic effect of SIN-1. OONO- itself was responsible for the positive inotropic effect, and appeared to modulate the activity of the sodium-calcium exchanger.

1. The cardiac effects of the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) were studied in frog fibres to evaluate the contribution of cyclic GMP-dependent mechanisms. 2. SNP and SNAP (0.1-100 microM) reduced the force of contraction in a concentration-dependent manner in atrial and ventricular fibres. This effect was associated with a reduction in the time to peak (TTP) and the time for half-relaxation of contraction (T). 3. SIN-1 (100 microM) also reduced the force of contraction in two-thirds of the atrial fibres. However, it exerted a positive inotropic effect in the remaining atrial fibres, as well as in most ventricular fibres. 4. The guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM) antagonized the negative inotropic effects of SIN-1 (50 microM) and SNAP (25 microM) but had no effect on the positive inotropic response to SIN-1 (100 microM). 5. In the presence of SIN-1, superoxide dismutase (SOD, 50-200 U ml-1) either potentiated the negative inotropic effect or turned the positive inotropic effect of the drug into a negative effect. SOD had no effects when applied alone or in the presence of SNAP. 6. 6-Anilino-5,8-quinolinedione (LY 83583, 3-30 microM), a superoxide anion generator also known as a cyclic GMP-lowering agent, exerted a positive inotropic effect, which was antagonized by SOD (200-370 U ml-1) but not by ODQ (10 microM). 7. We conclude that SNP, SNAP and SIN-1 exert cyclic GMP-dependent negative inotropic effects, which are attributed to the generation of NO. In addition, SIN-1 and LY 83583 exert cyclic GMP-independent positive inotropic effects, which require the generation of superoxide anion.

1. The effects of magnesium adenosine triphosphate (MgATP; also referred to as 'substrate') concentration on maximal force and shortening velocity have been studied at 5 C in single and thin bundles of striated muscle myofibrils. The minute diameters of the preparations promote rapid diffusional equilibrium between the bathing medium and lattice space so that during contraction fine control of substrate and product concentrations is achieved. 2. Myofibrils from frog tibialis anterior and rabbit psoas fast skeletal muscles were activated maximally by rapidly (10 ms) exchanging a continuous flux of pCa 8.0 for one at pCa 4.75 at a range of substrate concentrations from 10 microM to 5 mM. At high substrate concentrations maximal isometric tension and shortening velocity of both frog and rabbit myofibrils were very close to those determined in whole fibre preparations from the same muscle types. 3. As in frog and rabbit skinned whole fibres, the maximal isometric force of the myofibril preparations decreases as MgATP concentration is increased. The maximal velocity of unloaded shortening (V0) depends hyperbolically on substrate concentration. V0 extrapolated to infinite MgATP (3.6 +/- 0.2 and 0.8 +/- 0.03 l0 s-1 in frog and rabbit myofibrils, respectively) is very close to that determined directly at high substrate concentration. The Km is 210 +/- 20 microM for frog tibialis anterior and 120 +/- 10 microM for rabbit psoas myofibrils, values about half those found in larger whole fibre preparations of the same muscle types. This implies that measurements in whole skinned fibres are perturbed by diffusional delays, even in the presence of MgATP regenerating systems. 4. In both frog and rabbit myofibrils, the Km for V0 is about one order of magnitude higher than the Km for myofibrillar MgATPase determined biochemically in the same experimental conditions. This confirms that the difference between the Km values for MgATPase and shortening velocity is a basic feature of the mechanism of chemomechanical transduction in muscle contraction.

1. In the present work we have measured the pH of the secreted fluid within the gland lumen of isolated but intact gastric mucosa of Rana esculenta. Tissues were mounted in a double chamber allowing continuous perfusion of the mucosal and serosal compartment, and the measurements were made with double-barrelled pH glass microelectrodes inserted into the glands from the serosal surface under microscopic inspection. 2. During inhibition of H+ secretion by cimetidine (100 microM) the luminal gland pH (pHgl) averaged 7.60 +/- 0.05 pH units (mean +/- s.e.m.; n = 35), a value significantly higher than bath solution pH (7.45 +/- 0.02; P < 0.001) and also higher than intracellular pH of oxyntopeptic cells (pHi), which averaged 7.53 +/- 0.06 (n = 18). 3. Stimulation of acid secretion with histamine (500 microM) reversibly decreased pHgl to values which could be as low as 2.5. Together with electrophysiological criteria this response was routinely used to verify the proper location of the microelectrode tip within the gland lumen. 4. Stimulation with carbachol (100 microM) or pentagastrin (50 microM) in the presence of cimetidine rapidly and reversibly increased pHgl by 0.10 +/- 0.01 pH units (n = 24; P < 0.001) and 0.09 +/- 0.02 pH units (n = 6; P < 0.05), respectively. 5. The observation that gastric gland fluid is more alkaline than the bath solutions and that carbachol or pentagastrin further alkalinize it strongly suggests that oxyntopeptic cells participate in gastric alkaline secretion at least under cholinergic stimulation.

1. The distribution of L-type Ca2+ and tetrodotoxin-sensitive Na+ channels and of beta-adrenergic receptors was examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. Rod-shaped cells (250-300 microns long) were sealed at both ends to two patch-clamp pipettes and positioned transversally at different positions between the mouths of two microcapillaries separated by a thin wall. A combination of nifedipine (1 microM) and tetrodotoxin (0.3 microM) (blocking solution) was added to one capillary in order to inhibit macroscopic Ca2+ and Na+ currents (Ica and INa, respectively) in the part of the cell exposed to this capillary. 3. Moving the cell in 10-20 microns steps from the control capillary to the capillary containing the blocking solution induced step decreases in Ica and INa amplitudes. Complete block of both currents occurred when the entire cell was exposed to the blocking solution. 4. Each step decrease in current was due to the loss of activity of the functional Ca2+ and Na+ channels present in the slice of sarcolemmal membrane newly exposed to the blocking solution. These step current changes allowed longitudinal mapping of current density for Ca2+ and Na+ channels on the sarcolemmal membrane. 5. Addition of a submaximal concentration of isoprenaline (10 nM) to the control capillary induced a local increase in Ica which enabled examination of the distribution of functional beta-adrenergic receptors as well. 6. Our results demonstrate that Ca2+ and Na+ channels and beta-adrenergic receptors are equally and essentially uniformly distributed on the sarcolemmal of frog ventricular myocytes.

1. Force measurements in isolated myofibrils (15 degrees C; sarcomere length, 2.10 microns) were used in this study to determine whether sarcomeric proteins are responsible for the large differences in the amounts of active and passive tension of cardiac versus skeletal muscle. Single myofibrils and bundles of two to four myofibrils were prepared from glycerinated tibialis anterior and sartorius muscles of the frog. Skinned frog atrial myocytes were used as a model for cardiac myofibrils. 2. Electron microscope analysis of the preparations showed that: (i) frog atrial myocytes contained a small and variable number of individual myofibrils (from 1 to 7); (ii) the mean cross-sectional area and mean number of myosin filaments of individual cardiac myofibrils did not differ significantly from those of single skeletal myofibrils; and (iii) the total myofibril cross-sectional area of atrial myocytes was on average comparable to that of bundles of two to four skeletal myofibrils. 3. In maximally activated skeletal preparations, values of active force ranged from 0.45 +/- 0.03 microN for the single myofibrils (mean +/- S.E.M.; n = 16) to 1.44 +/- 0.24 microN for the bundles of two to four myofibrils (n = 9). Maximum active force values of forty-five cardiac myocytes averaged 1.47 +/- 0.10 microN and exhibited a non-continuous distribution with peaks at intervals of about 0.5 microN. The results suggest that variation in active force among cardiac preparations mainly reflects variability in the number of myofibrils inside the myocytes and that individual cardiac myofibrils develop the same average amount of force as single skeletal myofibrils. 4. The mean sarcomere length-resting force relation of atrial myocytes could be superimposed on that of bundles of two to four skeletal myofibrils. This suggests that, for any given amount of strain, individual cardiac and skeletal sarcomeres bear essentially the same passive force. 5. The length-passive tension data of all preparations could be fitted by an exponential equation. Equation parameters obtained for both types of myofibrils were in reasonable agreement with those reported for larger preparations of frog skeletal muscle but were very different from those estimated for multicellular frog atrial preparations. It is concluded that myofibrils are the major determinant of resting tension in skeletal muscle; structures other than the myofibrils are responsible for the high passive stiffness of frog cardiac muscle.

1. We have tested the widely accepted hypothesis that resting-state bicarbonate secretion of gastric fundus mucosa is mediated by Cl(-)-HCO3- exchange in the apical membrane of surface epithelial cells (SECs). To this end, SECs of isolated fundus mucosa of Rana esculenta were punctured with double-barrelled microelectrodes to measure intracellular pH (pHi). 2. No significant pHi changes were observed in response to changing luminal HCO3- and/or Cl- concentrations. The change in pHi (delta pHi) in response to luminal chloride substitution averaged 0.00 +/- 0.01 pH units (mean +/- S.E.M.; n = 48), and did not change after blocking putative basolateral acid/base transporters which could have masked the pHi response. 3. On the other hand, pHi responded readily and reversibly to luminal perfusion with either low-pH (pH 2.5) solution (delta pHi = -0.36 +/- 0.05; n = 4; P < 0.01) or CO2-free HCO3- Ringer solution (delta pHi = +0.10 +/- 0.01; n = 29; P < 0.001). These observations demonstrate that the solution change was effective and complete within 1 min and show that the apical membrane of SECs is permeable to CO2. 4. The apical membrane of frog SECs could not be stained with an antibody against the C-terminal end of the mouse Cl(-)-HCO3- exchanger isoform AE2, although this antibody readily stained the basolateral membrane of the oxyntopeptic cells (OCs). 5. In conclusion, the presence of a Cl(-)-HCO3- exchanger in the apical membrane of SECs of frog gastric fundus mucosa in the resting state could not be confirmed, but other models of HCO3- secretion cannot be fully excluded. Observations from electrical measurements, favouring a model of conductive HCO3- secretion, point to the OCs rather than the SECs as a site of origin of HCO3- secretion.

1. The time course of cross-bridge detachment-attachment following a step stretch was determined in single frog muscle fibres (at 4 degrees (1 and 2.1 microns sarcomere length) by imposing, under sarcomere length control by a striation follower, test step releases of various amplitudes (2-13 nm per half-sarcomere) at successive times (4-55 ms) after a conditioning stretch of approximately 4 nm per half-sarcomere. 2. The comparison with the control tension transients, elicited by releases not preceded by the conditioning stretch, shows that, early after the conditioning stretch, the quick tension recovery following small releases is depressed and the quick tension recovery following large releases is potentiated. Both effects are expected as a consequence of the strain produced in the cross-bridges by the conditioning stretch. 3. These effects disappear and the tension transient is reprimed, indicating substitution of freshly attached cross-bridges for strained cross-bridges, with a time constant of approximately 10 ms. 4. A novel multiple-exponential equation, based on the hypothesis of complete substitution of freshly attached cross-bridges for the cross-bridges that underwent the stretch, has been used to fit the whole tension transient following step stretches of different sizes (2-6 nm per half-sarcomere). For a stretch of 4 nm, the time constant of the exponential process responsible for cross-bridge detachment (tau d, 9.3 ms) almost coincides with the time constant of repriming as measured by the double-step experiments. The time constant of the exponential process representing the cumulative effects of attachment and force generation (tau 3) is 13.6 ms. 5. For stretches of different sizes the amount of quick tension recovery attributable to the reversal of the working stroke elicited by the stretches is estimated by subtracting, from the original tension transient, the contribution to tension recovery due to detachment-attachment of cross-bridges as estimated by the multiple-exponential analysis. Following this calculation, the structural change in the myosin heads responsible for the reversal of the working stroke can be 2 nm at maximum, suggesting that the elastic component in the cross-bridges is at least twice as rigid as previously thought.

1. The K+ channels of the apical membrane of the diluting segment (early distal tubule, EDT) of the frog are involved in the regulation of transepithelial NaCl transport. These channels are sensitive to pHi and intracellular Ca2+ (Ca2+i). Inhibition of transport by furosemide (frusemide) results in a compensatory increase in K+ channel activity. The aims of the present study were to determine whether pHi or Ca2+i were altered by furosemide, and to identify the means by which such changes were brought about. 2. Experiments were performed using single, microperfused EDT segments. Measurements of pHi and Ca2+i were made using the intracellular fluorescent probes, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and fura-2, respectively. 3. Furosemide increased pHi and Ca2+i. The intracellular alkalinization was the result of an alkaline shift in the set-point of the basolateral Na(+)-H+ exchanger. This response was dependent upon the increase in Ca2+i. 4. The increase in Ca2+i produced by furosemide was due to the release of Ca2+ from intracellular stores. Depletion of these stores, by 2,5-di-t-butylhydroquinone (TBQ) and caffeine, prevented the furosemide-induced changes in Ca2+ and pH. 5. Furosemide-induced activation of Na(+)-H+ exchange was prevented by the calmodulin antagonist, W-7. 6. Thus furosemide elicits a rise in Ca2+i which, via calmodulin, results in activation of Na(+)-H+ exchange. The resulting intracellular alkalinization would be expected to increase channel activity.

1. The effects of digoxin and ouabain on the calcium release flux from the sarcoplasmic reticulum (SR), isometric tension and intramembrane charge movement were studied in voltage clamped skeletal muscle fibres of the frog. 2. Both cardiac glycosides increased both calcium transients and simultaneously recorded tension at all membrane potentials, showing different effects on the peak and on the steady components of the calcium release flux. These effects were attained at an extracellular digoxin concentration of 5 nM and an estimated intracellular ouabain concentration of 1-2 nM. Digoxin and ouabain thus exerted their effects at the same concentration on calcium release in skeletal muscle as previously observed in isolated cardiac-type ryanodine receptor (RyR) calcium release channels. 3. The peak of SR calcium release increased at all voltages, with the largest potentiation at intermediate membrane potentials. This increase in calcium release flux was attained despite an unchanged SR calcium content. The attenuated release rate therefore reflected an increased number of open RyR channels rather than increased SR loading. 4. These effects could be attributed to an increase in calcium release activation and not a decrease in the rate of inactivation. Rather, the rate of inactivation was enhanced at all voltages as expected from the increased calcium concentration in the triadic junction. 5. In contrast, CMA (17 alpha-acetoxy-6-chloro-4, 6-pregnadiene-3,20-dione; 5 microM), a Na(+)-K(+)-ATPase inhibitor with no positive inotropic effects on the heart, neither influenced SR calcium release nor antagonized the effects of ouabain. 6. Both digoxin and ouabain preserved total intramembrane charge apart from a small negative shift in the mid-point voltage and increase in slope factor. 7. Both digoxin and ouabain induced calcium release from heavy SR vesicles at rates comparable to that induced by ryanodine or caffeine. 8. It is concluded that at least part of the inactivating component of SR calcium release involves distinct RyR calcium release channels that resemble the cardiac RyR isoform in its specific sensitivity to cardiac glycosides.

1. Single skeletal muscle fibres of Xenopus frogs were used to investigate the possibility that excitation-contraction (E-C) coupling can be impaired under conditions of elevated intracellular free Ca2+ ([Ca2+]i). 2. Fibres were stimulated with a train of up to 200 tetani at 10 or 20s intervals; this long-interval stimulation (LIS) scheme was chosen to minimize fatigue. After LIS, fibres were exposed to hypotonic Ringer solution for 5 min. At the end of LIS, force was about 90% of the original and the hypotonic challenge did not result in any force depression. 3. Caffeine, terbutaline and 2,5-di(tert-butyl)-1,4-benzohydroquinone increased both basal and tetanic [Ca2+]i. In ten out of thirteen fibres, the presence of any of these drugs during LIS resulted in a force reduction to about 10% of the control when fibres were returned to normal Ringer solution after the hypotonic challenge. Force production was severely depressed for at least 20 min and then recovered to control levels within 120 min. 4. Neither protease inhibitors nor a scavenger of reactive oxygen species prevented the impairment of E-C coupling. 5. It is concluded that after a period of elevated [Ca2+]i, E-C coupling in frog skeletal muscle becomes sensitive to the mechanical stress induced by exposure to hypotonic solution. The underlying molecular basis for this remains unclear.

1. The suction pipette technique was used to examine the effect of a conditioning pre-flash on the saturation time (tsat) of a bright test flash (intensity 10,000-250,000 isomerizations) delivered to intact salamander or toad rod outer segments. The conditioning flash was delivered 0-60 s before the test flash; its intensity was typically between six and sixty times dimmer than the test flash, and it was sufficient by itself to fully saturate the photocurrent. 2. A saturating pre-flash delivered before a saturating test flash reduced the tsat of the test flash. This was equivalent to a reduction in phototransduction gain (psi). 3. The pre-flash had little effect on tau zero the time constant of decay of the rate-limiting species in photoresponse inactivation (activated rhodopsin or the activated G-protein-phosphodiesterase complex). 4. The tsat declined exponentially as the separation time between a fixed intensity pre-flash and test flash was increased. The time constant (tau p) of decline in tsat was approximately 2.4s. The maximum reduction in tsat corresponded to a reduction in the apparent gain of phototransduction to approximately 0.10 of its original level. This exponential decline is consistent with a [Ca2+]i-mediated effect. 5. We conclude that the rate-limiting step in response inactivation and the step responsible for light-induced gain reduction constitute separate and distinct steps of the phototransduction cascade.

1. The activation of the L-type calcium current (ICa) was studied in normally polarized (-100 mV) cut skeletal muscle fibres of the frog with the double Vaseline-gap voltage-clamp technique. Both external and internal solutions were Ca2+ buffered. Solutions were made in order to minimize all but the Ca2+ current. 2. The voltage-dependent components of the time course of activation were determined by two procedures: fast and slow components were evaluated by multiexponential fitting to current traces elicited by long voltage pulses (5 s) after removing inactivation; fast components were also determined by short voltage pulses having different duration (0.5-70 ms). 3. The components of deactivation were evaluated after removing the charge-movement current from the total tail current by the difference between two short (50 and 70 ms) voltage pulses to 10 mV, moving the same intramembrane charge. Two exponential components, fast and slow (time constants, 6 +/- 0.3 and 90 +/- 7 ms at -100 mV; n = 26), were found. 4. The time onset of ICa was evaluated either by multiexponential fitting to the ICa activation or by pulses of different duration to test the beginning of the 'on' and 'off' inequality. This was at about 2 ms, denoting that it was very early. 5. The time constant vs. voltage plots indicated the presence of four voltage-dependent components in the activation pathway. Various kinetic models are discussed. Models with independent transitions, like a Hodgkin-Huxley scheme, were excluded. Suitable models were a five-state sequential and a four-state cyclic with a branch scheme. The latter gave the best simulation of the data. 6. The steady-state activation curve saturated at high potentials. It had a half-voltage value of 1 +/- 0.2 mV and the opening probability was only 0.82 +/- 0.2 at 20 mV (n = 32). This result implies a larger number of functional calcium channels than was previously supposed and is in agreement with the number of dihydropyridine (DHP) receptors calculated for the tubular system.

1. A fast perfusion system was used to analyse the kinetics of the response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.

1. The effects of acetylcholine (ACh) on the L-type Ca2+ current (ICa) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 microM Iso produced a half-maximal increase in ICa since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 microM ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only approximately 50%. 3. When 10 microM Iso or 30 microM Fsk were applied to a Ca(2+)-free solution on one half of the cell, ICa was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3-10 microM) to the remote part had no effect on ICa, while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates ICa by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of ICa does not involve any additional cAMP-independent mechanisms occurring downstream from cAMP generation.

1. In epithelial cells of frog skin, potassium ions are recycled across the basolateral membrane via an inward-rectifier, ATP-sensitive K+ channel (KATP channel). In this study, we show that aldosterone has a stimulatory effect on KATP channel activity and we have investigated the involvement of Na+-H+ exchange and intracellular pH (pHi) in this phenomenon. 2. Aldosterone (10 nM) produced an increase in the open probability of the KATP channel within 15 min from 0.21 +/- 0.05 to 0.93 +/- 0.10 (n = 8), measured in cell-attached patches. Aldosterone also increased the tolbutamide-sensitive K+ current across the basolateral membrane within 30 min from 17.2 +/- 1.9 to 30.3 +/- 1.6 microA cm-2 (n = 8) in nystatin-permeabilized whole skins. 3. The KATP channel is very sensitive to variations in cytosolic pH within the physiological range 7.0-7.4. 4. The intracellular pH of principal cells is regulated by Na+-H+ exchange, and the stimulatory effect of aldosterone on KATP channel activity was abolished by amiloride (100 microM) added on the basolateral side of the epithelium either before or after aldosterone treatment. 5. We propose that aldosterone activates the KATP channels via stimulation of Na+-H+ exchange. The rapidity of aldosterone activation of KATP channels is presented as evidence for a novel non-genomic steroid hormone effect on epithelial ion transport.

Isolated frog skin epithelium, mounted in an Ussing chamber and bathed in standard NaCl Ringer solution, recycles K+ across the basolateral membrane of principal cells through an inward-rectifier K+ channel (Kir) operating in parallel with a Na+-K+-ATPase pump. Here we report on the metabolic control of the Kir channel using patch clamping, short-circuit current measurement and enzymatic determination of cellular (ATP (ATPi). 2. The constitutively active Kir channel in the basolateral membrane has the characteristics of an ATP-regulated K+ channel and is now classed as a KATP channel. In excised inside-out patches the open probability (Po) of KATP channels was reduced by ATPi with half-maximum inhibition at an ATPi concentration of 50 microM. 3. ATPi measured (under normal Na+ transport conditions) with luciferin-luciferase was 1.50 +/- 0.23 mM (mean +/- S.E.M.; range, 0.4-3.3 mM n = 11). Thus the KATP channel would be expected to be inactive in intact cells if ATPi was the sole regulator of channel activity. KATP channels which were inactivated by 1 mM ATPi in excised patches could be reactivated by addition of 100 microM ADP on the cytosolic side. When added alone, ADP blocks this channel with half-maximal inhibition at [ADPi] > 5 mM. 4. Sulphonylureas inhibit single KATP channels in cell-attached patches as well as the total basolateral K+ current measured in frog skin epithelia perforated with nystatin on the apical side. 5. Na+-K+-ATPase activity is a major determinant of cytosolic ATP. Blocking the pump activity with ouabain produced a time-dependent increase in ATPi and reduced the open probability of KATP channels in cell-attached membranes. 6. We conclude that the ratio of ATP/ADP is an important metabolic coupling factor between the rate of Na+-K+ pumping and K+ recycling.

1. Whole-cell measurements were made from microvillous receptor neurones isolated from the frog vomeronasal organ. We examined the mechanisms that determined the value of the resting membrane potential. 2. Cells recorded in Ringer solution containing 4 mM K+ showed a resting membrane potential of -88 +/- 20 mV (mean +/- 1 S.D., n = 56). Sixty-six per cent of the cells had stable resting potentials more negative than the calculated equilibrium potentials for K+ (EK, -82 mV) indicating the presence of a hyperpolarizing outward pump current. 3. Cells recorded with an intracellular solution containing Na+ instead of K+, to set EK at 0 mV, presented stable membrane potentials in the range -65 to -119 mV when bathed in a normal Ringer solution. 4. Ouabain, a specific inhibitor of the Na+,K(+)-ATPase, blocked the outward sodium pump current (Ip) and depolarized the membrane. 5. The sodium pump current, measured as the current blocked by 0.5 mM dihydro-ouabain, was linearly related to the membrane potential in the range -60 to -120 mV. The reversal potential measured with a calculated free energy of ATP hydrolysis of -36.2 kJ mol-1 was estimated to be -143 mV. 6. Reduction of the external K+ concentration to 0 mM depolarized the membrane to less than -40 mV. Voltage-clamp observations in this condition indicated a reduction of Ip. Ouabain added to the bath reduced the blocking effect of low external K+. The addition of external K+ activated Ip and induced a rapid hyperpolarization of the cell membrane. 7. At membrane potentials more negative than -80 mV, an inward rectifying depolarizing current characterized as Ih was activated. When Ih was blocked by 5 mM external Cs+ the resting membrane potential increased. 8. These data indicate that the membrane potential of the vomeronasal receptor neurones is not generated by a passive diffusion of K+ ions but by the hyperpolarizing current created by the Na+,K(+)-ATPase. We propose that the resting potential is set by a balance between Ip and Ih. The physiological implications of these mechanisms for setting the resting potential are discussed.

1. The effect of the dynein inhibitor erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) on the osmotic water flow response to vasopressin or exogenous cAMP has been investigated in isolated toad urinary bladders. 2. Pretreatment with serosal EHNA had no effect on basal water flow, but inhibited the development and maintenance of the hydrosmotic response to vasopressin (20 mU ml-1) or 8-(4-parachlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8 CPT-cAMP; 0.1 mM). 3. The inhibitory effect of EHNA on vasopressin-induced water flow was dose dependent. Inhibition occurred in the dose range in which EHNA inhibits the ATPase and motor activities of dynein in vitro. 4. EHNA also inhibited the maintenance of the high rate of water flow established by prior exposure to vasopressin. 5. The inhibitory effect of EHNA on the onset phase of the vasopressin response was attenuated after exposure of the tissue to the microtubule-disruptive drug nocodazole but was fully additive with that of cytochalasin B. 6. EHNA inhibited basal and vasopressin-stimulated transepithelial sodium transport. 7. The findings support the view that EHNA inhibits hormone-induced water flow through an action on a cytoplasmic dynein. The results are consistent with the hypothesis that dynein is involved in the microtubule-based delivery of water channel-containing vesicles to the apical membrane of the granular epithelial cells during both the onset and maintenance of the water permeability response to vasopressin.

1. The force enhancement during and after stretch (0.15 micron per sarcomere) was studied during fused tetani of single fibres isolated from the anterior tibialis muscle of Rana temporaria (0.5-3.6 degrees C; sarcomere length, 2.05-2.65 microns). Changes in length were recorded simultaneously from the fibre as a whole (puller movement) and from marked segments (approximately 0.5 mm in length) of the same fibre. 2. The residual force enhancement after stretch (recorded at the end of a long tetanus) was found to be linearly related to the slow component of tension rise during the stretch ramp. 3. The fibres were released to shorten against a very small load at different times after stretch (load clamp). The shortening records derived after a preceding stretch exhibited a larger and steeper initial transient than that recorded in an isometric tetanus without stretch. The excess length change (LS; nanometres per half-sarcomere) recorded during the initial transient increased with the amplitude of stretch and was linearly related to the force enhancement produced by the stretch (FE; % of maximum tetanic tension) according to the following regression: LS = 0.200 FE + 8.65 (P < 0.001). The length changes recorded from the whole fibre agreed well with measurements from individual segments. 4. Slack-test measurements confirmed the existence of a large initial transient phase when the fibre was released to shorten after a preceding stretch. The excess length change determined from the slack tests agreed closely with the values derived from load-clamp recordings. 5. The results support the view that stretching a muscle fibre during tetanus leads to strain of elastic elements and, presumably, to variation of filament overlap due to non-uniform distribution of the length change within the fibre volume. Regions with greater filament overlap are likely to generate the long-lasting extra force referred to as 'residual force enhancement after stretch'. The elastic elements recruited during stretch can be presumed to play an essential part in this process by supporting regions in which the filament overlap has been reduced during the stretch ramp. Recoil of these elastic elements is responsible for the excess length change that is recorded during the initial transient after release as described under point 3.

1. Charge movement was studied in cut twitch fibres of Rana temporaria using a double Vaseline-gap voltage-clamp technique. 2. Replacement of Cl- by gluconate in the external solution reduced the magnitude of the early current component (I beta) substantially but affected the magnitude and slowed the kinetics of the hump current component (I gamma) slightly. 3. The early (Q beta) and hump (Q gamma) charge components in the gluconate solution were 11.8 +/- 2.3 and 88.0 +/- 4.8% (mean +/- S.E.M., n = 9), respectively, of those in the Cl- solution. 4. These results suggest that Q beta cannot be a precursor of Q gamma. Moreover, since fibres bathed in a gluconate solution can release calcium, Q beta is probably not involved in triggering calcium release.

1. Transient outward current was recorded in cultured frog melanotrophs with the whole-cell configuration of the patch-clamp technique. The ionic dependence, kinetics and pharmacological properties of the current were studied. The effects of the A1 adenosine receptor agonist R-N6-phenylisopropyl-adenosine (R-PIA) on this current were also investigated. 2. In tetrodotoxin- and cobalt-containing solution, depolarization from -120 mV elicited both transient and delayed outward currents. Pulses from -60 mV activated only a sustained late current. 3. 4-Aminopyridine (4 mM) reduced the transient outward current much more than the delayed outward current. In contrast, tetraethylammonium (10-20 mM) selectively reduced the delayed current. 4. Tail current measurements showed a positive shift in the reversal potential when external K+ concentration was increased, indicating that K+ was the predominant charge carrier. 5. Steady-state inactivation was complete at potentials positive to -10 mV and removed by hyperpolarization. 6. Inactivation of the transient current was slowed and accelerated in oxidizing and reducing conditions, respectively, confirming the involvement of an inactivating 'ball and chain' peptide. 7. R-PIA increased the transient current. The steady-state inactivation curve was shifted towards more positive potentials without changing the activation kinetics. Pretreatment with pertussis toxin (1 microgram ml-1) blocked the response to R-PIA. 8. It is concluded that frog melanotrophs possess an A-type current that is likely to play an important role in excitability. This current, which is directly modulated by A1 adenosine receptors through a Gi/G(o) protein, appears to be responsible for the inhibitory effects of adenosine on electrical activity.

1. L-type calcium currents were activated by depolarization of cut muscle fibres of the frog. The current was blocked by the dihydropyridine compound nifedipine (5-10 microM) and reactivated by flash photolysis of the drug. 2. In the presence of nifedipine, increasing the time interval between the onset of depolarization and the flash resulted in progressively faster kinetics of the flash-induced current. This change developed with a slow time course similar to that of normal current activation. 3. A fast gating mode of the normally slow L-type channel was induced by conditioning activation (500 ms prepulses) applied 80 ms before a test step to the same potential. After block by nifedipine, flash-photolysis was carried out 40 ms before the end of the long conditioning pulse. The flash-induced current had the same rapid time course as the current activated by the subsequent test voltage step. 4. Similarly, the time course of current activation was comparable for the voltage-induced fast mode activation (flash applied 5 ms before the test step) and the flash-induced activation 40 ms after the onset of the test depolarization. 5. Our data suggest that in frog skeletal muscle nifedipine inhibits calcium current activation by blocking a rapid channel gating step while the slow conformational change that normally limits the rate of activation of the L-type calcium channel remains unaffected. UV flash illumination results in a fast reactivation indicating that the channels need not be inactivated to be blocked by nifedipine.

1. The molecular mechanisms underlying pH regulation of skeletal muscle ATP-sensitive K+ (KATP) channels were studied using the patch clamp technique in the inside-out configuration. Two effects of intracellular protons were studied in detail: the decrease in magnitude of single-channel currents and the increase in open probability (Po) of nucleotide-inhibited channels. 2. The pH dependence of inward unit currents under different ionic conditions was in poor agreement with either a direct block of the pore by protons or an indirect proton-induced conformational change, but was compatible with the protonation of surface charges located near the cytoplasmic entrance of the pore. This latter electrostatic mechanism was modelled using Gouy-Chapman-Stern theory, which predicted the data accurately with a surface charge density of about 0.1 negative elementary charges per square nanometre and a pK (pH value for 50% effect) value for protonation of these charges of 6.25. The same mechanism, i.e. neutralization of negative surface charges by cation binding, could also account for the previously reported reduction of inward unit currents by Mg2+. 3. Intracellular alkalization did not affect Po of the KATP channels. Acidification increased Po. In the presence of 0.1 mM ATP (no Mg2+), the channel activation vs. pH relationship could be fitted with a sigmoid curve with a Hill coefficient slightly above 2 and a pK value of 6. This latter value was dependent on the ATP concentration, decreasing from 6.3 in 30 microM ATP to 5.3 in 1 microM ATP. 4. Conversely, the channel inhibition vs. ATP concentration curve was shifted to the right when the pH was lowered. At pH 7.1, the ATP concentration causing half-maximal inhibition was about 10 microM. At pH 5.4, it was about 400 microM. The Hill coefficient values remained slightly below 2. Similar effects were observed when ADP was used as the inhibitory nucleotide. 5. These results confirm that a reciprocal competitive link exists between proton and nucleotide binding sites. Quantitatively, they are in full agreement with a steady-state model of a KATP channel possessing four identical protonation sites (microscopic pK, 6) allosterically connected to the channel open state and two identical nucleotide sites (microscopic ATP dissociation constant, approximately 30 microM) connected to the closed state.

1. Passive force responses to ramp stretches at various velocities were measured in intact and skinned single muscle fibres isolated from the lumbricalis muscle of the frog. Force was measured using a fast capacitance transducer and sarcomere length was measured using a laser light diffraction technique at a point very close to the fixed end so as to avoid effects of fibre inertia. Experiments were performed at 15 degrees C with sarcomere length between 2.13 and 3.27 microns under high (170 mM) and low (20 mM) ionic strength. 2. The analysis shows that the force response is the sum of at least three components: (i) elastic (force proportional to the amount of stretch), (ii) viscous (force proportional to rate of stretch), and (iii) viscoelastic (resembling the response of a pure viscous element in series with an elastic element). 3. The amplitude of all these components increased progressively with sarcomere length in the whole range measured. 4. A further component, attributable to the short-range elasticity (SREC), was present in the force response of the intact fibres. 5. The amplitude of the force response decreased substantially upon skinning at high ionic strength but increased again at low ionic strength. The SREC was completely abolished by skinning. 6. None of the components of the force response was found to have the properties expected from the previously postulated 'weakly binding bridges'.

1. Force responses to ramp stretches were recorded in single muscle fibres isolated from the lumbricalis muscle of the frog. Stretches were applied at rest and at progressively increasing times after a single stimulus. 2. The increase of fibre stiffness that precedes tension development has a 'static' component that accounts for the whole fibre stiffness increase during the latent period and at very low tension at the beginning of the twitch. 3. Static stiffness increase was not affected by 2,3-butanedione-2-monoxime, a drug that almost completely inhibited twitch tension. 4. Static stiffness increased approximately 5-fold as the sarcomere length was increased from 2.1 to 2.84 microns. 5. These results suggest that static fibre stiffness increase is not attributable to the formation of non-force-generating cross-bridges.

1. The mechanism of transmitter release at the cytoneural junction of the frog posterior canal was investigated by recording intracellularly subthreshold postsynaptic potentials (EPSPs), and performing a statistical analysis of time intervals and peak amplitudes. In single units EPSPs display highly variable size, so it is not clear whether they are generated by the release of single quanta of transmitter and whether large ones represent giant events, multiquantal events, or the random summation of independent unitary events. 2. In units with low resting EPSP rates, peak amplitudes and time intervals between EPSPs were measured directly. Peak amplitude histograms were continuous, unimodal and well fitted by log normal distributions. Time-interval histograms were well described by single exponentials. 3. At high EPSP rates (either at rest or during experimental treatments), where single events overlapped extensively, peak amplitude histograms were skewed markedly towards high values. Under these conditions, the EPSP waveform was estimated by autoregressive fit to the autocorrelation of the recorded signal. The fit was used to build a Wiener filter, for sharpening the original signal, before computing time-interval and peak amplitude histograms. This yielded consistent log normal peak amplitude distributions with no 'excess' skewness, similar to those obtained with low resting rates. 4. After sharpening by the Wiener filter, shoulders or small second peaks in amplitude distributions were observed only at the highest EPSP rates (> 300 s-1). The number of 'multiquantal' events was reduced by Wiener filtering, and was in general consistent with the expectation that more than one independent event occurred within the duration of the single event. This suggests that the events are uniquantal, random and independent, i.e. miniature EPSPs (mEPSPs). 5. In general, peak amplitude distributions obtained with modified external Ca2+ concentration ([Ca2+]o) and/or during mechanical stimulation or under efferent activation were not significantly altered with respect to those obtained in the same units at rest. Time-interval histograms were generally mono-exponential at rest as well as during mechanical or efferent stimulation, and irrespective of [Ca2+]o. Resting mEPSP rate was slightly increased by elevated [Ca2+]o and reduced by low [Ca2+]o. The increase in mEPSP rate produced by mechanical excitation was depressed by both high and low [Ca2+]o, whereas both conditions enhanced mechanical inhibition. Efferent inhibition was little affected. High [Ca2+]o hastened adaptation during efferent facilitation. Low [Ca2+]o reduced peak response during facilitation, but suppressed its warning. 6. In the presence of ATP a consistent though transient increase in resting mEPSP rate was observed in about 50% of units.(ABSTRACT TRUNCATED AT 400 WORDS)

1. We compared the tension transient that follows a step change in sarcomere length in normal Ringer solution with that in Ringer solution made hypertonic by the addition of 98 mM sucrose. Steps were applied on tetanized single muscle fibres during either the isometric plateau or the steady force response to lengthening at low speed. Sarcomere length was controlled on selected fibre segments by a striation follower. Analysis is limited to phase 1 (the tension change simultaneous with the length step, mainly due to cross-bridge elasticity) and phase 2 (the quick phase of tension recovery, a manifestation of the cross-bridge elementary force-generating process). 2. At the isometric tetanus plateau the steady force is reduced by 19% in hypertonic solution, and the stiffness is slightly increased. During slow lengthening both steady force and stiffness are similar in normal solution and in hypertonic solution. In hypertonic solution the tension-to-stiffness ratio, a measure of the mean cross-bridge extension before the step, is markedly reduced in isometric conditions (-23%), but not during lengthening (-2%). 3. The plots of instantaneous tension versus the length change during the step show that in hypertonic medium the elasticity of the fibre is almost undamped. Thus the increase in stiffness cannot be attributed to an increase in viscosity. 4. In isometric conditions (T2-T1)/(Ti-T1), the proportion of the initial tension drop recovered at the end of phase 2, is not affected by hypertonicity for releases of moderate and large size (> 2 nm) and is reduced for small releases (< 2 nm) and for stretches. The abscissa intercept of the relation (T2-T1)/(Ti-T1) versus step amplitude is the same in both media. During lengthening, for releases of small and moderate size, (T2-T1)/(Ti-T1) is 20% lower in hypertonic solution. For large releases the slope of the relation is lower so that the abscissa intercept is not changed. 5. The speed of quick tension recovery following a step length change imposed in isometric conditions is slightly depressed in hypertonic solution. The relation between speed of recovery and step amplitude maintains its shape and is shifted downwards. During lengthening, the speed of quick tension recovery in hypertonic solution is less dependent on step amplitude than in normal solution, as if a more linear viscoelasticity is responsible for a large fraction of residual recovery.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Force transients in response to step perturbations in length were recorded in intact atrial cells from frog heart at various temperatures (6-15 degrees C). Length changes of various sizes and in either direction, complete in 0.5 ms, were applied to single myocytes near slack length (initial sarcomere length 2.1-2.2 microns) just before the peak of an isometric twitch. The frequency response of the force transducers used was 2-4 kHz in Ringer solution. 2. An early quick force recovery phase was clearly observed after the elastic force response to the length step and before the start of much slower recovery processes. The quick recovery phase became progressively faster with larger shortening steps and was almost as fast as that originally described in intact frog skeletal muscle fibres (rate constants above 1000 s-1 in large releases at 10 degrees C). 3. The force-extension relation determined at the end of the length change (T1 curve) indicates that an instantaneous shortening of 0.5-0.6% of the initial cell length (L0) brings the force to zero. The force--extension relation determined at the end of the quick recovery phase (T2 curve) showed that the early recovery leads to an almost complete restoration of the original force with small stretches and releases (up to 0.3% L0) and that it becomes negligible in shortening steps of about 1.4% L0. 4. The results suggest that the mechanical properties of attached cross-bridges and the rate of transitions between attached cross-bridge states are approximately the same in frog atrial cells and fast skeletal muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The mechanisms of K+ secretion into endolymph were studied on a preparation of isolated semicircular canal with different pharmacological inhibitors. Three periods of 5 or 30 min were performed, the first as control, the second in the presence of the drugs added to the apical or the basolateral bathing solution, and the third as recovery. Apical fluid was sampled at the beginning and the end of each period, transepithelial potential was recorded, Na+, K+, and Cl- concentrations, and K+ efflux, with 86Rb+ as a tracer, were measured and K+ fluxes were calculated. 2. When both sides of the epithelium were bathed with perilymph-like solution, the epithelium absorbed Na+, secreted K+, and generated a lumen positive potential. 3. The ATPases inhibitors, ouabain (10(-5) and 10(-3) M) and N-ethylmaleimide (10(-4) and 10(-3) M) inhibited the electrogenic K+ secretion when added to the basolateral fluid. N-ethylmaleimide (10(-3) M) applied to the apical fluid during a 5 min period decreased the K+ influx by 43% and the transepithelial potential by 66%. Other ATPase inhibitors, harmaline (10(-3) M), omeprazole (10(-4) M), vanadate (10(-4) M and 10(-3) M), N,N'-dicyclohexylcarbodiimide (DCC, 10(-5) M), 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl, 5 x 10(-6) M and 5 x 10(-5) M), and bafilomycin (10(-7) M) did not affect the K+ transport nor the transepithelial potential when they were added to the apical fluid. 4. The Na(+)-K(+)-Cl- co-transporter inhibitor, bumetanide, decreased both the transepithelial potential and the K+ transport when added to the basolateral solution but not to the apical one. At 10(-6) M, bumetanide maximally decreased the K+ influx by about 60%. 5. K+ channel blockers, quinine (10(-4) M), TEA (5 x 10(-3) M), added to the apical solution and barium (2 x 10(-3) M) added to either the apical or the basolateral solutions, did not affect the K+ transport and the transepithelial potential. 6. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) added to both apical and basolateral solutions did not affect the K+ transport and the transepithelial potential. 7. It is concluded that, in the ampulla of the semicircular canal, a basolateral Na(+)-K(+)-Cl- co-transporter energized by the Na+, K(+)-ATPase was involved for 60% in the K+ secretion into endolymph. The electrogenic K+ transport would partly depend on a N-ethylmaleimide-sensitive protein possibly located at the apical plasma membrane or intracellularly.

1. Perfused macropatch electrodes were used to stimulate and simultaneously measure release from two sites on the same terminal of the frog cutaneous pectoris muscle. 2. It was found that release occurring at one site often affected release at an adjacent site 50 microns away, either enhancing it ('spatial facilitation') or depressing it ('spatial depression'). Spatial facilitation (or depression) was defined as the release produced by a test pulse at the second site (test electrode) when preceded by a pulse at the first site (prepulse electrode) divided by the release produced by the test pulse alone. 3. Spatial facilitation varied with the time interval between the prepulse and the test pulse. Peak spatial facilitation, which on the average was 2.14, occurred with an interval of 1-3 ms. With longer intervals spatial facilitation decayed with a time constant between 3-6 ms. When the time interval between the prepulse and the test pulse was zero (no delay), the release after the test pulse was always depressed. 4. When Ca2+ was omitted from the perfusate of the prepulse electrode, spatial facilitation was abolished. When a brief hyperpolarizing pulse followed the depolarizing prepulse with zero delay spatial facilitation was also abolished. 5. Electrotonic spread or Ca2+ diffusion within the axon terminal are excluded as coupling agents for spatial facilitation. It is suggested that the coupling agent may possibly be related to a hypothetical release-promoting factor.

1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose-dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (-)-Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF-DX 116 >> pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Ca2+ inward currents were measured by voltage clamping cut skeletal muscle fibres of the frog (Rana esculenta) in a double-Vaseline-gap system. 2. In order to study the basis of the previously described fast gating mode induced in the Ca2+ inward current by a conditioning depolarization we quantitatively analysed the response to differing features of the conditioning prepulse. 3. The faster activation seen during the second of two depolarizations was confined to the component of the inward current which could be blocked by 5 to 10 microM nifedipine. 4. By applying depolarizing conditioning pulses of gradually increasing length the time course of the transition to the fast gating mode could be determined. 5. Both the transition to the fast gating mode (point 4) caused by a depolarization and the slow inward current activated during the same depolarization showed similar voltage-dependent kinetics. 6. The kinetic change of the test current appeared to be equal when the same fractional activation was achieved at the end of the conditioning pulse independent of its duration or amplitude. 7. Flash photolysis of nifedipine in the interval between conditioning and test pulse showed that the predepolarization causes a rate-enhancing effect even though the slow channels were blocked by nifedipine during the conditioning pulse. 8. We conclude that the transition of the calcium channel from its slow to its fast gating mode is determined by the slow voltage-dependent reaction which limits the rate of channel opening under control conditions. This reaction is apparently not prevented by the binding of nifedipine and the block of current flow through the channel.

1. Apparatus for recording the small tensions developed by electrically stimulated single intact myocytes of frog heart is described. A laser-light optoelectronic transducer was used. The compliance of the force probes was 10-20 nm/nN, with a frequency response of 600-900 Hz in Ringer solution. The myocyte shortening during an ordinary twitch contraction was no greater than 1% of the slack length. The overall sensitivity of the transducer system was 5-10 mV/nN, with a total noise of 0.5-1 nN peak to peak. The experiments were performed at 20-23 degrees C on either atrial or ventricular myocytes at 2.15-2.2 microns sarcomere length, in 1 mM-Ca2+ Ringer solution. 2. Isoprenaline (5 microM), increases in external Ca2+ concentration ([Ca2+]o), and shortening of stimulus interval potentiated the myocyte twitch tension. The dependence of twitch characteristics on these inotropic interventions for all the atrial and ventricular myocytes tested was comparable to that of multicellular preparations under similar experimental conditions. This implies that the enzymatic isolation procedure had not altered the physiological properties of the myocytes. 3. The stimulus interval-tension relation for premature twitches of atrial and ventricular myocytes showed (i) a very steep rising phase in the region of intervals just longer than 0.52 and 0.66 s (the duration of the mechanical refractoriness in atrial or ventricular cells), (ii) a peak, at intervals of 0.7-0.8 s, where the twitch tension was strongly potentiated compared to that of the controls, and (iii) as the stimulus interval was further increased, a progressive return to the control level. The stimulus interval-tension relation for steady-state conditions exhibited similar characteristics. 4. The degree of tension potentiation by isoprenaline was greater in the controls than in the earliest test twitches. The result was that the stimulus interval-tension relations for isoprenaline-treated myocytes showed a gentler rise and a lower peak than for untreated cells. 5. The stimulus interval-tension relation of the heart is a basic property of its cells. It is related to changes in the activation level. The higher the activation level reached in control twitches, the lower the stimulus interval-dependent potentiation capability.