Rapamycin: clinical results and future opportunities.
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Animals↗
Biomedical subjects
J S Camardo
Publications and source records attributed to J S Camardo.
Rapamycin (sirolimus, rapamune).
Rapamycin is a novel immunosuppressive agent that is undergoing clinical trials for use in allograft rejection therapy. This paper reviews its in-vitro biological properties, the current state of knowledge concerning its mechanism of action, and its therapeutic applications.
Animals↗
Comparison of the serotonin-sensitive and Ca(2+)-activated K+ channels in Aplysia sensory neurons.
1. Single potassium channel currents were recorded in cell-attached and cell-free patches from Aplysia sensory neurons. Two prominent classes of K+ channels were identified that have similar single-channel current amplitude at 0 mV: (1) the resting conductance serotonin-sensitive K+ channels (S-channels) previously described in these neurons; and (2) a calcium-activated K+ channel. A series of experiments were carried out which enable these channels to be distinguished on the basis of their biophysical properties. These experiments also provide further insight into the gating and ionic selectivity of the S-channel. 2. In inside-out patches, single calcium-activated K+ channel currents (IK,Ca) show a linear i-V curve with a slope conductance of 66 pS (normal sea water outside, 360 mM-KCl inside) whereas single S-channels display an outwardly rectifying i-V curve with a slope conductance of 90 pS at 0 mV. 3. The gating of IK,Ca has a steep voltage dependence, with open probability showing an e-fold increase for a 16 mV depolarization. Increasing internal calcium concentration from 0.2 to 10 microM shifts the activation curve by 60 mV in the hyperpolarizing direction. 4. S-channel gating is independent of internal calcium (from less than 10 nM up to 100 microM). Steady-state open probability of the S-channel generally shows a weak dependence on membrane potential, with open probability increasing twofold for a 30-100 mV depolarization. Occasional patches were observed with S-channels displaying a much greater voltage sensitivity, with open probability increasing e-fold for a 16-20 mV depolarization. 5. S-channels are selective for K+ over Na+. The selectivity ratio depends on the ratio of Na+ to K+ concentration on the same side of the membrane. Increasing K+ concentration appears to increase relative Na+ permeability, suggesting ion-ion interactions within the channel. 6. We conclude that Aplysia sensory neurons contain two prominent distinguishable classes of K+ channels, the Ca(2+)-independent S-channel and a Ca(2+)-activated channel. The gating properties of the S-channels allow them to contribute outward repolarizing current over a wide range of membrane potentials so that their modulation by neurotransmitters contributes to changes in both resting potential and action potential duration.
Animals↗
Single sodium channels from canine ventricular myocytes: voltage dependence and relative rates of activation and inactivation.
1. Single sodium channel currents were recorded from canine ventricular myocytes in cell-attached patches. The relative rates of single-channel activation vs. inactivation as well as the voltage dependence of the rate of open-channel inactivation were studied. 2. Ensemble-averaged sodium currents showed relatively normal activation and inactivation kinetics, although the mid-point of the steady-state inactivation (h infinity) curve was shifted by 20-30 mV in the hyperpolarizing direction. This shift was due to the bath solution, which contained isotonic KCl to depolarize the cell to 0 mV. 3. Steady-state activation showed less of a voltage shift. The threshold for eliciting channel opening was around -70 mV and the mid-point of activation occurred near -50 mV. 4. The decline of the ensemble-averaged sodium current during a maintained depolarization was fitted by a single exponential function characterizing the apparent time constant of inactivation (tau h). The apparent rate of inactivation was voltage dependent, with tau h decreasing e-fold for a 15.4 mV depolarization. 5. The relative contributions of the rates of single-channel activation and inactivation in determining the time course of current decay (tau h) were examined using the approach of Aldrich, Corey & Stevens (1983). Mean channel open time (tau o) showed significant voltage dependence, increasing from 0.5 ms at -70 mV to around 0.8 ms at -40 mV. At -70 mV tau h was much greater than tau o, while at -40 mV the two time constants were similar. 6. The degree to which the kinetics of single-channel activation contribute to tau h was studied using the first latency distribution. The first latency function was fitted by two exponentials. The slow component was voltage dependent, decreasing from 19 ms at -70 mV to 0.5 ms at -40 mV. The fast component (0.1-0.5 ms) was not well resolved. 7. Comparing the first latency distribution with the time course of the ensemble-averaged sodium current at -40 mV showed that activation is nearly complete by the time of peak inward sodium current. However, at -70 mV, activation overlaps significantly with the apparent time course of inactivation of the ensemble-averaged current. 8. Using the methods of Aldrich et al. (1983) we also measured the apparent rate of open-channel closing (a) and open-channel inactivation (b). Both rates were voltage dependent, with a showing an e-fold decrease for an 11 mV depolarization and b showing an e-fold increase for a 30 mV depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Regulation of single potassium channels by serotonin in the cell bodies of the tail mechanosensory neurons of Aplysia californica.
Sensory neurons in the pleural ganglion of Aplysia mediate the afferent portion of the tail withdrawal reflex. Previous work has shown that in these neurons and in the siphon sensory neurons of Aplysia, serotonin modulates a steady-state non-inactivating potassium current called the S current. Using the technique of patch clamping, we have examined the kinetics of single potassium channels and found that they share the properties of the S potassium channel of the siphon sensory neurons. This channel has an elementary slope conductance of 73 +/- 9.98 pS (mean +/- S.E.M.) and shows Goldman rectification. It is active at the resting potential and does not inactivate with maintained depolarization. Bath application of serotonin in a majority of experiments decreased the functional number of channels in the patch.
Animals↗
Modulation of the 'S' K+ channel by cAMP-dependent protein phosphorylation in cell-free membrane patches.
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Animals↗
Modulation of the serotonin-sensitive potassium channel in Aplysia sensory neurone cell body and growth cone.
Using single-channel recording, we have been able to obtain some insight into the molecular mechanism of a modulatory transmitter action in Aplysia sensory neurones. Our results show that serotonin produces a slow EPSP and increases action potential duration in the sensory neurones by producing prolonged closures of the S potassium channel. Such closures appear to be mediated by cyclic AMP-dependent phosphorylation of a membrane protein which may be the channel. Modulation of S channels by serotonin also occurs in sensory neurone growth cones. This provides the first direct evidence that channel modulation occurs in nerve processes and increases the likelihood of channel modulation at the nerve terminal.
Animals↗
Serotonin and cyclic adenosine 3':5'-monophosphate modulate the potassium current in tail sensory neurons in the pleural ganglion of Aplysia.
Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdrawal reflex in Aplysia californica undergo heterosynaptic facilitation of transmitter release during sensitization. As in the siphon sensory neurons, the transmitter serotonin produces facilitation and also elicits a slow, decreased conductance excitatory postsynaptic potential (EPSP) in these neurons. Using voltage clamp and biochemical analyses, we have found that the slow EPSP in the pleural sensory neurons is due to a decrease in a potassium conductance identical to the S potassium current characterized in siphon sensory neurons. Like the S current, the current modulated by serotonin in the pleural sensory neurons is a non-inactivating potassium current, and it contributes to both the resting and action potentials. The current reverses in 120 mM external K+ at -20 mV, close to the predicted Nernst equilibrium potential. Intracellular cesium blocks the serotonin response, but the current is not blocked by equimolar substitution of barium for calcium, nor by 50 mM tetraethylammonium chloride. The effect of serotonin is cAMP dependent, since serotonin elevates cAMP and both cAMP injection and forskolin mimic the serotonin response. These results indicate that the mechanism associated with sensitization of the siphon-gill withdrawal reflex, a slow decreased potassium conductance, is also a component of the neuronal circuitry underlying modulation of another reflex, the tail withdrawal reflex. Therefore, two distinct populations of neurons subserving similar behavioral functions have related biophysical and biochemical properties.
Aplysia↗
Two endogenous neuropeptides modulate the gill and siphon withdrawal reflex in Aplysia by presynaptic facilitation involving cAMP-dependent closure of a serotonin-sensitive potassium channel.
We have found that two endogenous neuropeptides in Aplysia, the small cardioactive peptides SCPA and SCPB, facilitate synaptic transmission from siphon mechano-sensory neurons and enhance the defensive withdrawal reflex that these sensory neurons mediate. Single-channel recording revealed that these peptides close a specific K+ channel, the S channel, which is sensitive to cAMP. Moreover, the peptides increase cAMP levels in these sensory neurons. This reduction in K+ current slows the repolarization of the action potential in these cells, which increases transmitter release. In these actions, the SCPs resemble both noxious sensitizing stimuli, which enhance the reflex, and serotonin. Bioassay of HPLC fractions of abdominal ganglion extracts and immunocytochemistry indicate that both the SCPs and serotonin are present in the ganglion and are found in processes close to the siphon sensory neurons, suggesting that these transmitters may be involved in behavioral sensitization. Recent evidence suggests that one group of identified facilitatory interneurons, the L29 cells, does not appear to contain either the SCPs or serotonin but may use yet another facilitatory transmitter. Thus, it appears that several transmitters can converge to produce presynaptic facilitation in the sensory neurons of the defensive withdrawal reflex. All of the transmitters studied here, the SCPs and serotonin, act via an identical molecular cascade: cAMP-dependent closure of the S-K+ channel, broadening of the presynaptic action potential, and facilitation of transmitter release.
Action Potentials↗
Differential emergence of cellular mechanisms mediating habituation and sensitization in the developing Aplysia nervous system.
The development of the cellular substrates underlying habituation and sensitization, two simple forms of learning, was examined at a polysynaptic sensory-to-motor connection in the neural circuit mediating defensive mucus release in the marine mollusc, Aplysia californica. Animals were studied throughout juvenile life, stages 9 (40 days of development) to 12 (95 days), and into adulthood, stage 13 (120 days), starting just after metamorphosis when mucus release first becomes evident. Homosynaptic depression, which mediates habituation, was already present in its adult form in stage 9. Heterosynaptic facilitation, which mediates sensitization, appeared in stage 10 and reached maturity during stages 11 and 12. Thus, the development of synaptic plasticity in this circuit occurs in discrete phases in which the gradual emergence of heterosynaptic facilitation occurs only after homosynaptic depression is well established.
Aging↗
Modulation of a specific potassium channel in sensory neurons of Aplysia by serotonin and cAMP-dependent protein phosphorylation.
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Animals↗
Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurones.
We have identified a serotonin-sensitive K+ channel with novel properties. The channel is active at the testing potential; its gating is moderately affected by membrane potential and is not dependent on the activity of intracellular calcium ions. Application of serotonin to the cell body or intracellular injection of cyclic AMP causes prolonged and complete closure of the channel, thereby reducing the effective number of active channels in the membrane. The closure of the channel can account for the increases in the duration of the action potential, Ca2+ influx, and transmitter release which underlie behavioural sensitization, a simple form of learning.
Animals↗
Human ventricular refractoriness: effects of increasing current.
The ventricular effective refractory period is commonly employed as a measurement of ventricular excitability. Because the current strength used to make this determination varies among laboratories, the relation of refractoriness and current was examined over a range of current strengths from 0.1 to 10 mA. Sixty determinations of refractoriness at variable current strengths were made in 40 patients using the extrastimulus technique with a rectangular pulse of 1 ms duration. These data were obtained by measuring the effective refractory period at threshold current and at 0.25 to 0.50 mA increments from threshold up to 10 mA. In these studies the drive stimulus (S1) and extrastimulus (S2) were kept at the same amplitude. In all patients the ventricular effective refractory period decreased as the current increased. The total decrease ranged from 8 to 100 ms (mean +/- standard deviation 36.9 +/- 17.1). The current strength at which the ventricular effective refractory period became fixed (that is, less than 2 ms change in ventricular effective refractory period with further increase in current strength) varied among the patients, but in all instances equaled or exceeded 1.8 mA, which in all but three patients was greater than three times threshold. The curves relating current strength and refractoriness were shifted to the left at shorter cycle lengths with no change in threshold. These data suggest that (1) current strength-effective refractory period curves more completely characterize ventricular excitability than does a ventricular effective refractory period at single current strength; and (2) studies of drug effects, alterations of autonomic tone, or reentrant arrhythmias, which may affect or are affected by ventricular refractoriness, may be enhanced by more complete measurements of refractoriness afforded by the current strength-effective refractoriness curves.
Adolescent↗
Strength-interval relation in the human ventricle: effect of procainamide.
The effects of procainamide on strength-interval relations were evaluated in 18 patients. At plasma concentrations of 4.3 to 13.6 micrograms/ml procainamide had minimal effects on threshold current in late diastole, but in early diastole it shifted the strength-interval curve to the right. The basic strength-interval relation (that is, decreasing refractory period as current is increased) was not altered. The control refractory period decreased by a mean of 44 ms as the current was increased from threshold to 10 mA, whereas a mean decrease of 42 ms was observed after procainamide. However, the steep portion of the strength-interval curve(absolute refractory period) was shifted to longer coupling intervals by a mean value of 24 ms. These findings suggest that procainamide may primarily affect active membrane properties, but exert little net effect on passive membrane properties late in diastole.
Action Potentials↗
Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of rat.
The proposal of the subfornical organ (SFO) as a site of receptors for drinking induced by angiotensin II (AII) was investigated with several mutually confirmatory experiments. Intracranial injections of physiological doses of AII elicited drinking if and only if applied directly to the SFO (Experiment I). Ablation of the SFO selectively (Experiment 2) and permanently (Experiment 4) eliminated drinking elicited by physiological doses of intravenously infused AII. Animals in which SFO had been ablated responded normally to cellular dehydration but reduced responding to the extracellular thirsts of beta-adrenergic activation and hyperoncotic colloid dialysis (Experiment 3). Infusion of saralasin, an AII antagonist, directly into the SFO selectively and reversibly antagonized intravenous AII drinking (Experiment 5). The hypothesis that the SFO contains dipsogenic receptors for circulating AII is strongly supported.
Angiotensin II↗
The dipsogenic potency of peripheral angiotensin II.
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Angiotensin II↗
Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K+ channels of Aplysia sensory neurones in cell-free membrane patches.
Selected actions of neurotransmitters and hormones on ion channels in nerve and muscle cells are now thought to be mediated by cyclic AMP-dependent protein phosphorylation. Although the cyclic AMP-dependent protein kinase (cAMP-PK) affects the cellular properties of several neurones, its mode of action at the single-channel level has not been characterized. In addition, little is known about the identity or subcellular localization of the phosphoproteins that control channel activity and, in particular, whether the critical substrate proteins are cytoplasmic or membrane-associated. In Aplysia sensory neurones, serotonin produces a slow modulatory synaptic potential mediated by cAMP-PK that contributes to presynaptic facilitation and behavioural sensitization. Previously, we have found that serotonin acts on cell-attached membrane patches to produce prolonged all-or-none closures of a specific class of K+ channels (S channels) whose gating is weakly dependent on voltage and independent of intracellular calcium. We demonstrate here that in cell-free membrane patches from Aplysia sensory neurones, the purified catalytic subunit of cAMP-PK produces all-or-none closures of the S channel, simulating most (but not all) aspects of the action of serotonin on cell-attached patches. This result suggests that protein kinase acts on the internal surface of the membrane to phosphorylate either the channel itself or a membrane-associated protein that regulates channel activity.
Adenosine Triphosphate↗