Energetics of sodium transport in toad urinary bladder.
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The ratio of the rate of transepithelial sodium transport, JNa, across the isolated toad urinary bladder to the simultaneously measured rate of transport-dependent metabolism, JsbCO2, has been measured as a function of the transepithelial electrical voltage, deltapsi. The ratio remains constant with a mean value of 18 to 20 over the range of imposed voltages of 0 to +70 mV. With increasing hyperpolarization of the bladder, JNa decreases and the calculated electromotive force or apparent "ENa" of the sodium pump increases. From thermodynamic and kinetic arguments it is shown that the apparent "ENa" approaches the maximal electrochemical potential gradient, ENa, against which sodium can be transported by this tissue only when JNa approximately 0. At this unique condition F ENa (in which F is the Faraday constant) is the maximal free energy of the chemical reaction driving sodium transport and thus equal to the maximal extramitochondrial phosphorylation potential and the maximal free energy of the mitochondrial respiratory chain within the transporting cells. (+info)
Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).
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The effect of a conditioning depolarizing current pulse (80-200 micros) on quantal secretion evoked by a similar test pulse at another site was examined in visualized motor-nerve terminal branches of amphibian endplates (Bufo marinus). Tetrodotoxin (200 nM) and cadmium (50 microM) were used to block voltage-dependent sodium and calcium conductances. Quantal release at the test electrode was depressed at different distances (28-135 microm) from the conditioning electrode when the conditioning and test pulses were delivered simultaneously. This depression decreased when the interval between conditioning and test current pulses was increased, until, at an interval of approximately 0.25 ms, it was negligible. At no time during several thousand test-conditioning pairs, for electrodes at different distances apart (28-135 microm) on the same or contiguous terminal branches, did the electrotonic effects of quantal release at one electrode produce quantal release at the other. Analytic and numerical solutions were obtained for the distribution of transmembrane potential at different sites along terminal branches of different lengths for current injection at a point on a terminal branch wrapped in Schwann cell, in the absence of active membrane conductances. Solutions were also obtained for the combined effects of two sites of current injection separated by different time delays. This cable model shows that depolarizing current injections of a few hundred microseconds duration produce hyperpolarizations at approximately 30 microm beyond the site of current injection, with these becoming larger and occurring at shorter distances the shorter the terminal branch. Thus the effect of a conditioning depolarizing pulse at one site on a subsequent test pulse at another more than approximately 30 microm away is to substantially decrease the absolute depolarization produced by the latter, provided the interval between the pulses is less than a few hundred microseconds. It is concluded that the passive cable properties of motor nerve terminal branches are sufficient to explain the effects on quantal secretion by a test electrode depolarization of current injections from a spatially removed conditioning electrode. (+info)
How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2+ release in amphibian pacemaker cells.
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1. The mechanism by which sympathetic transmitters increase the firing rate of pacemaker cells was explored in isolated cells from the sinus venosus of the cane toad Bufo marinus. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 and membrane potential and currents were recorded with the nystatin perforated-patch technique. 2. Adrenaline or isoprenaline (2 microM) increased the transient rise in [Ca2+]i and increased the firing rate; these effects were blocked by propranolol (2 microM). 3. To determine whether the changes in [Ca2+]i might influence the firing rate we studied agents which affect either the loading or the release of Ca2+ from the sarcoplasmic reticulum (SR). Rapid application of caffeine (10 mM) to spontaneously firing cells caused a large Ca2+ release from the SR and the cells were then quiescent for 24 s. In the presence of beta-adrenergic stimulation the caffeine-induced [Ca2+]i was 14 % larger but the period of quiescence after application was reduced to 12 s. 4. Ryanodine, at either low (1 microM) or high (> 10 microM) concentration, stopped firing. However, when the SR store content of Ca2+ was tested with caffeine, at low ryanodine concentration the SR Ca2+ store was empty whereas at the high concentration the SR store was still loaded with Ca2+. beta-Adrenergic stimulation was not able to restore firing at the low concentration of ryanodine but did restore firing at the high ryanodine concentration. 5. An SR Ca2+ pump blocker, 2, 5-di(tert-butyl)-1,4-hydroquinone (TBQ) which depletes the SR store of Ca2+, also rapidly and reversibly stopped spontaneous firing. 6. The relation between the amplitude of the [Ca2+]i transient and firing rate established in the presence of ryanodine was similar when firing was restored by beta-stimulation. 7. In both spontaneously firing and voltage-clamped cells, depleting the SR store with either ryanodine or TBQ suggested that about half of the Ca2+ which contributes to the calcium transient is released from the SR. 8. These results show that the amplitude of the [Ca2+]i transient is an important factor in the firing rate of toad pacemaker cells and consequently agents which modify SR Ca2+ release influence firing rate. The effects of beta-stimulation on firing rate seem to be largely mediated by changes in amplitude of the [Ca2+]i transient. (+info)
Variability in the time course of single photon responses from toad rods: termination of rhodopsin's activity.
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We examined the responses of toad rod photoreceptors to single photons of light. To minimize the effects of variability in the early rising phase, we selected sets of responses that closely matched the rise of the mean single photon response. Responses selected in this way showed substantial variations in kinetics, appearing to peel off from a common time course after different delays. Following incorporation of the calcium buffer BAPTA, the time to peeling off was retarded. Our analysis indicates that it is not necessary to invoke a long series of reaction steps to explain the shutoff of rhodopsin activity. Instead, our results suggest that the observed behavior is explicable by the presently known shutoff reactions of activated rhodopsin, modulated by feedback. (+info)
Expression of membrane transporters in cane toad Bufo marinus oocytes.
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Membrane transport proteins (transporters and ion channels) have been extensively expressed in amphibian oocytes. The aims of this study were to determine whether oocytes from the cane toad Bufo marinus could be used as an alternative expression system to the broadly used Xenopus laevis oocytes. mRNAs encoding plasma membrane transporters NaSi-1 and sat-1 (sulphate transporters), NaDC-1 (dicarboxylate transporter), SGLT-1 (Na(+)/glucose cotransporter) and rBAT and 4F2 hc (amino acid transporters) were injected into B. marinus oocytes. All led to significant induction of their respective transport activities. Uptake rates were comparable with those in X. laevis oocytes, with the exception of rBAT, which was able to induce amino acid uptake only in X. laevis oocytes, suggesting that rBAT may require an endogenous X. laevis oocyte protein that is absent from B. marinus oocytes. Transport kinetics were determined for the NaSi-1 cotransporter in B. marinus oocytes, with identical results to those obtained in X. laevis oocytes. NaSi-1 specificity for the Na(+) cation was determined, and the anions selenate, molybdate, tungstate, oxalate and thiosulphate could all inhibit NaSi-1-induced sulphate transport. This study demonstrates that cane toad oocytes can be used successfully to express plasma membrane proteins, making this a viable heterologous system for the expression of proteins. (+info)
Distribution of active protein kinase C in smooth muscle.
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To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction. (+info)
The role of calcium stores in fatigue of isolated single muscle fibres from the cane toad.
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1. Intracellular calcium ([Ca2+]i) and tension were measured from single muscle fibres dissected from the cane toad (Bufo marinus). The amount of Ca2+ which could be released from the sarcoplasmic reticulum (SR) was estimated by brief (approximately 20 s) exposures to 4-chloro-m-cresol (4-CmC) or caffeine. 2. Muscle fatigue was produced by repeated tetani at 4 s or shorter intervals and continued until tension had fallen to 50% of the control. The intracellular free calcium concentration during a tetanus (tetanic [Ca2+]i) first increased and then steadily declined to 43+/-2% of control by the time tension had fallen to 50%. Over the period of fatigue the rapidly releasable Ca2+ from the SR fell to 46+/-6% of control. Tension and tetanic [Ca2+]i recovered to 93+/-3% and 100+/-4% of the control values after 20 min of rest. Over the same period rapidly releasable SR Ca2+ recovered to 98+/-12%. 3. When a similar number of tetani (200) were repeated at longer intervals (10 s), fibres showed only a small reduction in tension (to 85+/-1%) and tetanic [Ca2+]i did not change significantly. Under these conditions the rapidly releasable SR Ca2+ did not change significantly. 4. The recovery of rapidly releasable SR Ca2+ after fatigue was unaffected by removal of extracellular calcium but did not occur when oxidative phosphorylation was inhibited with cyanide. 5. These results suggest that an important cause of the decline of tetanic [Ca2+]i during fatigue is an equivalent decline in the amount of rapidly releasable SR Ca2+. The results show that the decline of rapidly releasable SR Ca2+ is related to a metabolic consequence of fatigue and are consistent with the hypothesis that Ca2+ precipitates with phosphate in the SR during fatigue. (+info)
Glycogen content and excitation-contraction coupling in mechanically skinned muscle fibres of the cane toad.
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1. Mechanically skinned skeletal muscle fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the relationship between fibre glycogen content and fibre capacity to respond to transverse tubular (T-) system depolarization. 2. A large proportion of total fibre glycogen remained in mechanically skinned muscle fibres exposed to aqueous solutions. This glycogen pool (about 80% of total fibre glycogen) was very stable when the preparation was incubated in a rigor solution (pH 7.0) but decreased gradually at a rate of 0.59+/-0.20% min-1 in a relaxing solution (200 nM [Ca2+]). The rate was considerably higher (2.66+/-0.38% min(-1)) when the preparations were exposed to 30 microM [Ca2+]. An even greater rate of glycogen loss was found after T-system depolarization-induced contractions. The Ca2+-dependent loss of fibre glycogen was caused by endogenous glycogenolytic processes. 3. Silver stained SDS gels of components eluted into relaxing solution from single skinned fibres revealed a rapid (2 min) loss of parvalbumin and at least 10 other proteins varying in molecular mass between 10 and 80 kDa but there was essentially no loss of myosin heavy and light chains and actin. Subsequent elution for a further 30 min in either relaxing or maximally Ca2+-activating solution did not result in additional, appreciable detectable loss of fibre protein. 4. Depletion of fibre glycogen was associated with loss of fibre ability to respond to T-system depolarization even though the bathing solutions contained high levels of ATP (8 mM) and creatine phosphate (10 mM). 5. The capacity of mechanically skinned fibres to respond to T-system depolarization was highly positively correlated (P<0.0001) with initial fibre glycogen concentration. 6. In conclusion, the results show that (i) the capacity of skeletal muscle to respond to T-system depolarization is related directly or indirectly to the non-washable glycogen pool in fibres, (ii) this relationship holds for conditions where glycogen is not required as a source of energy and (iii) the mechanically skinned fibre preparation is well suited to study the regulation of endogenous glycogenolytic enzymes. (+info)