Single Exponential Time Constant Research Articles

"Delayed" endocytosis is regulated by extracellular Ca2+ in snake motor boutons.

When cooled below approximately 7 degrees C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This "delayed" endocytosis following release from cold block provides an opportunity to study clathrin-dependent endocytotic mechanisms in temporal isolation from those events, such as Ca2+ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35 degrees C. Moreover, the rate of endocytosis varied significantly with change in [Ca2+]o; the rate at 7.2 mM (single exponential time constant, approximately 3 s) was approximately double that at 0 mM (single exponential time constant, approximately 7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca2+]o, potentially regulated by changes in the milieu of the synaptic cleft during neural activity.

'Delayed' endocytosis is regulated by extracellular Ca2+ in snake motor boutons

When cooled below ≈7 °C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This ‘delayed’ endocytosis following release from cold block provides an opportunity to study clathrin-dependent endocytotic mechanisms in temporal isolation from those events, such as Ca2+ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35 °C. Moreover, the rate of endocytosis varied significantly with change in [Ca2+]o; the rate at 7.2 mM (single exponential time constant, ≈3 s) was approximately double that at 0 mM (single exponential time constant, ≈7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca2+]o, potentially regulated by changes in the milieu of the synaptic cleft during neural activity.

Effects of membrane potential and tension on prestin, the outer hair cell lateral membrane motor protein.

1. Under whole-cell voltage clamp, the effects of initial voltage conditions and membrane tension on gating charge and voltage-dependent capacitance were studied in human embryonic kidney cells (TSA201 cell line) transiently transfected with the gene encoding the gerbil protein prestin. Conformational changes in this membrane-bound protein probably provide the molecular basis of the outer hair cell (OHC) voltage-driven mechanical activity, which spans the audio spectrum. 2. Boltzmann characteristics of the charge movement in transfected cells were similar to those reported for OHCs (Q(max) = 0.99 +/- 0.16 pC, z = 0.88 +/- 0.02; n = 5, means +/- S.E.M.). Unlike that of the adult OHC, the voltage at peak capacitance (V(pkcm)) was very negative (-74.7 +/- 3.8 mV). Linear capacitance in transfected cells was 43.7 +/- 13.8 pF and membrane resistance was 458 +/- 123 Mohms. 3. Voltage steps from the holding potential preceding the measurement of capacitance-voltage functions caused a time- and voltage-dependent shift in V(pkcm). For a prepulse to -150 mV, from a holding potential of 0 mV, V(pkcm) shifted 6.4 mV, and was fitted by a single exponential time constant of 45 ms. A higher resolution analysis of this time course was made by measuring the change in capacitance during a fixed voltage step and indicated a double exponential shift (tau(0) = 51.6 ms, tau(1) = 8.5 s) similar to that of the native gerbil OHC. 4. Membrane tension, delivered by increasing pipette pressure, caused a positive shift in V(pkcm). A maximal shift of 7.5 mV was obtained with 2 kPa of pressure. The effect was reversible. 5. Our results show that the sensitivity of prestin to initial voltage and membrane tension, though present, is less than that observed in adult OHCs. It remains possible that some other interacting molecular species within the lateral plasma membrane of the native OHC amplifies the effect of tension and prior voltage on prestin's activity.

Subthreshold inactivation of Na+ and K+ channels supports activity-dependent enhancement of back-propagating action potentials in hippocampal CA1.

Back-propagating action potentials in CA1 pyramidal neurons may provide the postsynaptic dendritic depolarization necessary for the induction of long-term synaptic plasticity. The amplitudes of back-propagating action potentials are not all or none but are limited in amplitude by dendritic A-type K+ channels. Previous studies of back-propagating action potentials have suggested that prior depolarization of the dendritic membrane reduces A-type channel availability through inactivation, resulting in an enhanced, or boosted, dendritic action potential. However, inactivation kinetics in the subthreshold potential range have not been directly measured. Furthermore, the corresponding rates of Na+ channel inactivation with depolarization have not been considered. Here we report in cell-attached patches (150-220 microm from the soma, 32 degrees C) that at 20-mV positive to rest, A-type K+ channels inactivated with a single exponential time constant of 6 ms, whereas Na+ channels inactivated with a time constant of 37 ms. The ratio of available Na+ to K+ current increased as the duration of the depolarization increased. Thus the subthreshold properties of Na+ and A-type K+ channels provide a mechanism by which information about the level of synaptic activity may be encoded in the amplitude of back-propagating action potentials.

Two types of pharmacologically distinct Ca(2+) currents with voltage-dependent similarities in zona fasciculata cells isolated from bovine adrenal gland.

Voltage-activated Ca(2+) currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca(2+): 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba(2+), 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: -40, 0 and +60 mV, respectively. The two components, fully inactivated at -10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about -15 and -34 mV, respectively. The sustained component was larger in Ba(2+) than in Sr(2+) and Ca(2+). Ni(2+) (20 microm) selectively blocked the transient component while Cd(2+) (10 microm) selectively blocked the sustained one. (+/-)Bay K 8644 (0.5 microm) increased the sustained component and nitrendipine (0.5-1 microm) blocked it selectively. The sustained component was inhibited by calciseptine (1 microm). Both components were unaffected by omega-conotoxin GVIA and MVIIC (0.5 microm). These results show that two distinct populations of Ca(2+) channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells.

Characterization with barium of potassium currents in turtle retinal Müller cells.

Müller cells are highly permeable to potassium ions and play a crucial role in maintaining potassium homeostasis in the vertebrate retina. The potassium current found in turtle Müller cells consists of two components: an inwardly rectifying component and a linear, passive component. These currents are insensitive to broadband potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP) and well blocked by barium. Differential block by the polyamine spermine suggests that these currents flow through different channels. In this study, we used barium ions as a probe to investigate the properties of these currents by whole cell, voltage-clamp recordings from isolated cells. Current-voltage (I-V) relationships generated from current responses to short (35 ms) and long (3.5 s) voltage pulses were fit with the Hill equation. With extracellular barium, the time course of block and unblock was voltage and concentration dependent and could be fit with single exponential functions and time constants larger than 100 ms. Blocking effects by extracellular barium on the two types of currents were indistinguishable. The decrease of the outward current originates in part due to charge effects. We also found that intracellular barium was an effective blocker of the potassium currents. The relative block of the inward rectifier by intracellular barium suggests the existence of two "apparent" binding sites available for barium within the channel. Under depolarizing conditions favoring the block by internal polyamines, the Hill coefficient for barium binding was 1, indicating a single apparent binding site for barium within the pore of the passive linear conductance. The difference in the steepness of the blocking functions suggests that the potassium currents flow through two different types of channels, an inward rectifier and a linear passive conductance. Last, we consider the use of barium as an intracellular K(+) channel blocker for voltage-clamp experiments.

Evidence of a cerebrovascular postarteriole windkessel with delayed compliance.

A pronounced temporal mismatch was observed between the responses of relative cerebral blood volume (rCBV) measured by magnetic resonance imaging and relative cerebral blood flow measured by laser-Doppler flowmetry in rat somatosensory cortex after electrical forepaw stimulation. The increase of relative cerebral blood flow after stimulus onset and decrease after stimulus cessation were accurately described with a single exponential time constant of 2.4 +/- 0.8 seconds. In contrast, rCBV exhibited two distinct and nearly sequential processes after both onset and cessation of stimulation. A rapid change of rCBV (1.5 +/- 0.8 seconds) occurring immediately after onset and cessation was not statistically different from the time constant for relative cerebral blood flow. However, a slow phase of increase (onset) and decrease (cessation) with an exponential time constant of 14 +/- 13 seconds began approximately 8 seconds after the rapid phase of CBV change. A modified windkessel model was developed to describe the temporal evolution of rCBV as a rapid elastic response of capillaries and veins followed by slow venous relaxation of stress. Venous delayed compliance was suggested as the mechanism for the poststimulus undershoot in blood oxygen-sensitive magnetic resonance imaging signal that has been observed in this animal model and in human data.

Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse.

1. Simultaneous patch clamp and rapid microfluorometric calcium measurements were performed on sixty-five motoneurones in slices of the nucleus hypoglossus in the brainstem of 2- to 6-day-old mice. 2. Hypoglossal motoneurones were particularly vulnerable to mechanical or metabolic stress during isolation of in vitro slice preparations. Therefore, experimental conditions were optimized for functional integrity, as judged by spontaneous rhythmic activity of hypoglossal nerves (XII). 3. Calcium concentrations in the cell soma were monitored with a temporal resolution in the millisecond time domain during depolarizing voltage steps. Ratiometric fluorescence measurements were made using a rapid monochromator (switching tau < 10 ms), a photomultiplier tube and the calcium sensitive dyes fura-2 and mag-fura-5. 4. Dynamics of somatic calcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients were approximated to a single exponential component and decay time constants showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0.7 +/- 0.2 s, suggesting rapid somatic calcium dynamics under physiological conditions. 5. By a process of back-extrapolation, the 'added buffer' method, a calcium binding ratio of 41 +/- 12 (9 cells) was obtained indicating that 98% of the calcium ions entering a hypoglossal motoneurone were bound by endogenous buffers. 6. Endogenous calcium binding ratios in hypoglossal motoneurones were small compared with those of other neurones with comparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium-related excitotoxicity might partially result from low concentrations of calcium buffers in these cells.

The pro-convulsant actions of corticotropin-releasing hormone in the hippocampus of infant rats

Whole-cell patch-clamp and extracellular field recordings were obtained from 450- μm-thick brain slices of infant rats (10–13 days postnatal) to determine the actions of corticotropin-releasing hormone on glutamate- and GABA-mediated synaptic transmission in the hippocampus. Synthetic corticotropin-releasing hormone (0.15 μM) reversibly increased the excitability of hippocampal pyramidal cells, as determined by the increase in the amplitude of the CA1 population spikes evoked by stimulation of the Schaffer collateral pathway. This increase in population spike amplitude could be prevented by the corticotropin-releasing hormone receptor antagonist α-helical (9–41)-corticotropin-releasing hormone (10 μM). Whole-cell patch-clamp recordings revealed that, in the presence of blockers of fast excitatory and inhibitory synaptic transmission, corticotropin-releasing hormone caused only a small (1–2 mV) depolarization of the resting membrane potential in CA3 pyramidal cells, and it did not significantly alter the input resistance. However, corticotropin-releasing hormone, in addition to decreasing the slow afterhyperpolarization, caused an increase in the number of action potentials per burst evoked by depolarizing current pulses. Corticotropin-releasing hormone did not significantly change the frequency, amplitude or kinetics of miniature excitatory postsynaptic currents. However, it increased the frequency of the spontaneous excitatory postsynaptic currents in CA3 pyramidal cells, without altering their amplitude and single exponential rise and decay time constants. Corticotropin-releasing hormone did not change the amplitude of the pharmacologically isolated (i.e. recorded in the presence of GABA A receptor antagonist bicuculline) excitatory postsynaptic currents in CA3 and CA1 pyramidal cells evoked by stimulation of the mossy fibers and the Schaffer collaterals, respectively. Current-clamp recordings in bicuculline-containing medium showed that, in the presence of corticotropin-releasing hormone, mossy fiber stimulation leads to large, synchronized, polysynaptically-evoked bursts of action potentials in CA3 pyramidal cells. In addition, the peptide caused a small, reversible decrease in the amplitude of the pharmacologically isolated (i.e. recorded in the presence of glutamate receptor antagonists) evoked inhibitory postsynaptic currents in CA3 pyramidal cells, but it did not significantly alter the frequency, amplitude, rise and decay time constants of spontaneous or miniature inhibitory postsynaptic currents. These data demonstrate that corticotropin-releasing hormone, an endogenous neuropeptide whose intracerebroventricular infusion results in seizure activity in immature rats, has diverse effects in the hippocampus which may contribute to epileptogenesis. It is proposed that the net effect of corticotropin-releasing hormone is a preferential amplification of those incoming excitatory signals which are strong enough to reach firing threshold in at least a subpopulation of CA3 cells. These findings suggest that the actions of corticotropin-releasing hormone on neuronal excitability in the immature hippocampus may play a role in human developmental epilepsies.

L-type calcium current activation in cultured human myotubes.

The time course of activation of the skeletal muscle L-type calcium channel was studied in voltage-clamped myotubes derived from human satellite cells. The slow L-type current was isolated by inactivating faster calcium current components using appropriate prepulses or by subtracting the currents not blocked by 5 microM nifedipine. The L-type current exhibited a single exponential activation and time constants which showed little voltage dependence in the range +10 to +50mV. Currents blocked by nifedipine could be partially restored by UV-light flash photolysis. When a flash of light was applied during a depolarizing step, the activation time course of the resulting inward current contained a rapid, almost instantaneous component followed by a slower component. The amplitude of the rapid component was different when the flash was applied at different times during the depolarizing step: depolarization first increased and then decreased the fraction of channels which could rapidly be restored from the block by photolysis. Plotted versus time after the onset of the depolarization this fraction closely matched the time course of the L-type current obtained before the block by nifedipine. This indicates that the slow gating recations of the Ca2+ channel remain functional in the nifedipine-blocked state. Large conditioning depolarizations which had been shown to enhance the speed of L-type current activation in frog muscle fibres showed no effect in human myotubes. Numerical simulations using a gating scheme proposed for frog muscle demonstrate that such differences can be caused by changing just a single kinetic parameter.

Properties of spontaneous inhibitory synaptic currents in cultured rat spinal cord and medullary neurons

1. To identify the type(s) and properties of inhibitory postsynaptic receptor(s) involved in synaptic transmission in cultured rat embryonic spinal cord and medullary neurons, we have used whole cell patch-clamp techniques to record miniature inhibitory postsynaptic currents (mIPSCs) in the presence of tetrodotoxin, DL-2-amino-5-phosphonovaleric acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. 2. The mIPSCs recorded from both spinal cord and medullary neurons had skewed amplitude distributions. 3. The glycinergic antagonist strychnine and the GABAergic antagonist bicuculline each decreased both the frequency and mean peak amplitudes of mIPSCs. We conclude that both glycine and gamma-aminobutyric acid (GABA) are neurotransmitters at inhibitory synapses in our cultured cells. 4. Most (approximately 96-97%) mIPSCs decay with single-exponential time constants, and decay time distributions were consistently best fitted by the sum of four Gaussians with decay constants as follows: D1 = 5.8 +/- 0.1 (SE) ms (n = 63), D2 = 12.2 +/- 0.2 ms (n = 61), D3 = 23.2 +/- 0.4 ms (n = 54), and D4 = 44.7 +/- 1.0 ms (n = 57). We conclude that the four classes of decay times represent kinetically different inhibitory postsynaptic receptor populations. 5. Strychnine and bicuculline usually had one of two different effects on the mIPSC decay time constant distributions; either selective decreases in the frequency of mIPSCs with decay times in certain classes (i.e., the D1 class was reduced by bicuculline, the D2 class by strychnine, and the D3 and D4 classes by both antagonists) or a nonselective depression in the frequency of mIPSCs with decay times in all four classes. The particular effect observed in a given neuron was correlated with the presence or absence of ATP and guanosine 5'-triphosphate (GTP) in the patch pipette. Namely, in 71% of the antagonist applications where the pipette contained ATP and GTP, the result was a nonselective decrease in mIPSCs in all decay time constant classes. Conversely, in 54% of the antagonist applications in their absence, the result was a selective decrease in the frequency of mIPSCs in specific decay time constant classes. 6. In some experiments, mIPSCs reappeared in antagonist solution after an essentially complete block. Recovery from block in the continued presence of antagonist was never observed in the absence of ATP and GTP (8 neurons), and, at the same time, 5 of 9 neurons patched with ATP and GTP in the pipette did show recovery (56%).

Cardiodynamic improvement following PGE 1 administration in sheep with congestive heart failure

The cardiodynamic effects of Prostaglandin E 1 were studied in sheep with congestive heart failure. Six Merino ewes were equipped surgically with high fidelity micromanometers and left ventricular pressures measured. Heart rate, left ventricular systolic and end-diastolic pressures, [ +dP dt ]max and a single-exponential time constant (T) were calculated on a computer from the recorded pressure curves and the Wilcoxon signed rank test used to determine the statistical significance of the differences. Prostaglandin E 1 (20 μg.kg −1) significantly improved left ventricular contractility and relaxation rate as seen respectively from [ +dP dt ]max and the single-exponential time-constant and reduced the afterload conditions under which the heart operated, during various stages of congestive heat failure, without significant tachycardia during the more advanced stages of the disease. The results show that [ +dP dt ]max increased between 16% (stage 4) and 38% (stage 1); T shortened between 14% (stage 4) and 25% (stage 1) and left ventricular systolic pressure decreased between 14% (stage 4) and 21% (stage 1) when prostaglandin E 1 was administered during four stages of left ventricular pump failure, which ranged from a 20% decrease in [ +dP dt ]max (congestive heart failure stage 1) to a 50% decrease in [ +dP dt ]max (congestive heart failure stage 4). In contrast to normal animals, prostaglandin E 1 caused an increase in left ventricular end-diastolic pressure during the more advanced stages of pump failure. Nevertheless the favourable cardiodynamic changes brought about by prostaglandin E 1 administration might contribute, together with afterload reduction, to a general haemodynamic improvement during congestive heart failure.

Calcium-dependent sodium current in the marine ciliateEuplotes vannus

Ca and Na inward currents were recorded upon depolarizations in Euplotes after the blockage of K outward currents with intracellular Cs ions. The Na current was analyzed under voltage clamp and had the following properties: it activated to a maximum within 150 msec and partly inactivated during sustained voltage steps. It had a positive equilibrium potential between 25 and 30 mV and could be carried by Na or Li ions but not by K, choline or Tris ions. The current revealed a prominent associated inward tail current which deactivated with a single-exponential time constant of 118 msec. Both the current and its tail were strongly reduced after reduction of the extracellular Na concentration. Externally applied K channel blocker tetraethylammonium chloride did not block the current. Either EGTA injection into the cell or nonlethal deciliation with ethanol eliminated the current and its tail. These results indicate the existence of a Na conductance within the membrane of Euplotes which is activated by the intracellular level of free Ca2+.

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules.

Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP. Pf was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75 mM inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3 +/- 0.1 sec (SEM, seven preparations, 23 degrees C), corresponding to a Pf of 5 x 10(-4) cm/sec; the activation energy (Ea) for Pf was 17.6 +/- 0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised approximately 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6 +/- 2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membranes) and 0.190 (microsomes), and were unaffected by VP. We conclude: (1) granules have among the lowest water permeabilities of biological membranes, (2) granule water permeability is not altered by bladder pretreatment with VP, (3) granule membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.

Solvent Polarity Dependent Photophysics of a Fixed‐Distance, Symmetric Chlorophyll Dimer. A Model of the Special Pair in Photosynthetic Reaction Centers

AbstractThe synthesis and properties of a fixed‐distance symmetric bis‐pyrochlorophyllide‐a molecule are described. The molecule, 1, consists of two methyl pyrochlorophyllide‐a (MePChlide‐a) moieties which share a common vinyl group at the 2‐position of each macrocycle. The two chlorophylls are trans to one another across the vinyl linkage. The dihedral angle between the plane of each macrocycle and the vinyl linkage is about 50°. The Qy absorption band of this dimer occurs at 689 nm and is essentially independent of solvent. The band is red‐shifted by about 27 run relative to that of MePChlide‐a. The fluorescence maximum of 1 occurs at 722 nm. This is a red‐shift of 49 nm relative to methyl MePChlide‐a. The circular dichroism spectrum of 1 shows strong, nonconservative bands in the red region. The fluorescence quantum yield, fluorescence lifetimes, and picosecond transient absorbance kinetics all exhibit a very strong dependence on solvent polarity. The fluorescence quantum yields of 1 in toluene, butyronitrile, and N, N‐dimethyl formamide (DMF) are 0.18, 0.008, and 0.002, respectively. The bleach of the 689 nm band (following excitation with a 1.5‐ps, 610‐nm laser pulse) recovers with single exponential time constants of 2.51 ns, 100 ps, and 64 ps in toluene, butyronitrile, and DMF, respectively. Possible mechanisms for the polarity‐dependent increase of the nonradiative decay rate of 1 are considered.

Time course of systolic loading is an important determinant of ventricular relaxation.

We studied the dependency of left ventricular relaxation on the timing of an abrupt increase in systolic load. In 10 canine isolated heart-lung preparations, a load step of 15 mmHg was imposed at specific intervals throughout systole, and the time of loading was defined as the interval from the R wave to the completion of the load step (R-load interval). Preload was held constant. The right atrium was paced at a cycle length of 450 ms. The decay of left ventricular pressure during isovolumic relaxation was described by a single exponential time constant (Texp). Load effects on isovolumic relaxation were expressed as a percent change in Texp as compared with Texp of the beat preceding the load intervention. Loads imposed early in systole consistently prolonged Texp [mean delta Texp = +17.01 +/- 1.64% (SE) for R-load intervals less than or equal to 120 ms]. Load changes late in systole consistently abbreviated Texp [mean delta Texp = -9.49 +/- 0.86% (SE) for R-load intervals greater than or equal to 130 ms]. The transition from augmentation to diminution of Texp always occurred when the R-load interval was 120-130 ms. The mean time interval of electromechanical systole for the test beats was not significantly different (P greater than or equal to 0.05) from that of the control beats [R-load intervals less than or equal to 120: test = 247.0 +/- 27.8 (SD) ms; control = 246.6 +/- 26.8 (SD) ms] [R-load intervals greater than or equal to 130: test = 243.3 +/- 29.1 (SD) ms; control = 243.8 +/- 28.4 (SD) ms].(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular sodium and lithium NMR relaxation times in the perfused frog heart

We have used a combination of a shift reagent and mathematical filtering or presaturation of the extracellular sodium resonance for the quantitative investigation of the intracellular sodium and lithium relaxation times in the perfused frog heart. While the T1 of the intracellular sodium was found to consist of a single-exponential time constant (approximately 23 ms), the T2 was better fit as a double-exponential decay with time constants of approximately 2 and 17 ms. However, the relative amplitudes of the two time constants in the T2 decay were found to be inconsistent with those which would be expected from a homogeneous pool of nuclei undergoing quadrupolar interactions. The relaxation times were not changed by a fivefold increase in the intracellular sodium level (due to perfusion with a ouabain-containing buffer). The T1 and T2 of the intracellular lithium (after perfusion with lithium-containing buffer) were both well fit by single exponentials (700- and 31-ms time constants, respectively).

Calcium‐activated potassium channels in rat muscle inactivate from a short‐duration open state.

Single channel recording techniques were applied to the study of activation and inactivation of Ca2+-activated K+ channels in excised patches of membrane from rat muscle grown in culture. The concentration of intracellular surface Ca2+ was 0.6 microM in all experiments. The time course of the averaged open probability during depolarizing voltage steps of 1 s duration was biphasic for steps more positive than 20 mV; a rapid activation phase was followed by a much slower apparent inactivation with a single exponential time constant in the range of 400-800 ms. The peak open probability and degree of inactivation increased as the steps were made more positive (+30 to +80 mV) or the holding potential more negative (+30 to -40 mV). A conditional probability analysis of the open intervals immediately adjacent to the long-duration shut intervals resulting from inactivation revealed that transitions to and from the inactivated state occurred almost exclusively via a short-duration open state (mean lifetime less than 200 microseconds). The rate of transition from the short-duration open state to the inactivated state was rapid (typical rate constant 1879/s) and was sufficiently probable that as many as one out of every three short-duration openings were followed by inactivation. Normal (non-inactivating) closure of the channel from this open state was also rapid (rate constant 4003/s). At constant voltage (+50 mV) and Ca2+ (0.6 microM), the channel opened to the short-duration open state approximately every 75 ms, suggesting that the slow inactivation of the averaged open probability might have been limited in part by the rate at which the channel entered the short-duration open state.

Subpicosecond time resolved multiphoton ionization: Excited state dynamics of cis-stilbene under collision free conditions

Subpicosecond time resolved multiphoton ionization is demonstrated to be a useful technique for probing unimolecular dynamics in vapor phase samples. Vapor phase cis-stilbene molecules are observed to decay out of their initially prepared S1 state with a single exponential time constant of 0.32 ps. This decay, thought to correspond to a rapid twisting of the molecule about the ethylenic double bond, occurs in roughly one c=c double bond torsional vibrational period.

Effects of the ATP analog 5′-adenylyl methylenediphosphonate on acid secretion in frog gastric mucosa

1. 1. The ATP analog 5′-adenylyl methylenediphosphonate (AMP-PCP) added to the serosal bathing solution of the chambered bullfrog gastric mucosa inhibits acid secretion, raises the transepithelial potential difference, and causes an increase in lag time before resumption of secretion after anoxia. These effects are similar in direction and magnitude to those caused by ATP. 2. 2. While AMP-PCP stimulates the production of inosine in this tissue, it is apparently not itself a precursor for inosine, and seems to act by stimulating catabolism of endogenous purines. 3. 3. AMP-PCP is not a substrate for adenosine deaminase or AMP deaminase, has no marked effects on mitochondrial respiration or phosphorylation, and is not catabolised by gastric mucosa. 4. 4. Washout kinetics of AMP-PCP loaded tissues show this material to be exiting with a single exponential time constant of about 10 min. By contrast, inosine shows 2 washout exponentials of 5 and 30 min. The appropriate analysis suggests that the cells may be more permeable to AMP-PCP than to inosine. 5. 5. It is suggested that the inhibitory action of AMP-PCP (and by extension, ATP and ADP) is due to the possession by these compounds of a nitrogen with an unshared pair of electrons, as has been suggested for other secretory inhibitors.