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P F Baker

Publications and source records attributed to P F Baker.

At least 91 records · Page 5Linked to original sources
PubMed1973

Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons.

1. The Ca-sensitive photoprotein aequorin was injected into squid axons and the light response to stimulation or depolarizing voltage clamp pulses recorded.2. The effects of Mn(2+), Co(2+), Ni(2+), La(3+) and of the organic Ca antagonists D-600 and iproveratril on the early tetrodotoxin-sensitive and late tetrodotoxin-insensitive components of the light response were studied.3. The late tetrodotoxin-insensitive component can be blocked, reversibly, by concentrations of Mn, Co and Ni that reduce but do not block the tetrodotoxin-sensitive component. The late component can also be blocked by La(3+) and the organic Ca antagonists D-600 and iproveratril.4. Mn(2+), Co(2+), Ni(2+) and the drug D-600 all reduce the Na currents, but have little effect on either outward or inward K currents. Tetraethylammonium blocks the outward K current but has no appreciable effect on the tetrodotoxin-insensitive entry of Ca.5. Concentrations of Mn between 5 and 50 mM substantially reduce the light output during a train of action potentials; they also slightly reduce the rate of rise of the action potential.6. On pharmacological grounds it is concluded that the tetrodotoxin-insensitive component of Ca entry does not represent Ca ions passing through the K permeability channels. There must exist a potential-dependent late Ca channel that is distinct from the well known Na and K channels of the action potential. A possible function for this late Ca channel in the coupling of excitation to secretion is discussed.

Action Potentials↗
PubMed1973

Calcium entry in response to maintained depolarization of squid axons.

1. Intracellular aequorin was used to monitor changes in Ca entry in response to maintained depolarization either produced electrically or by exposure to K-rich solutions.2. External K concentrations greater than 50 mM produce a phasic light response. The light rises to a peak in a few sec and then falls in 0.5-5 min to a new steady level that is always greater than the level in the absence of K.3. The phasic light response does not result from depletion of available aequorin at the periphery of the axon, but rather seems to reflect a phasic entry of Ca in response to depolarization.4. Similar phasic responses are produced by prolonged electrical depolarization. These results are consistent with depolarization serving both to activate and also to inactivate Ca entry.5. Following inactivation and after return to normal sea-water, there is an appreciable relative refractory period during which the response both to K-rich sea-water and electrical depolarization is reduced in size. Complete recovery takes 10-15 min.6. The response to 410 mM-KCl is dependent on the previous treatment of the preparation. Pre-treatment with 100 or 200 mM-KCl reduced the response to 410 mM-KCl. The potential for half inactivation was about -25 mV in 112 mM-Ca and -40 mV in 20 mM-Ca.7. The rate of onset of inactivation is potential dependent and is faster for depolarizations to zero potential than for smaller ones.8. The phasic Ca entry produced by K-rich solutions is insensitive to external tetrodotoxin and internal tetraethylammonium ions, but is blocked by external Mn(2+), Co(2+) and Ni(2+) ions and by the drugs D-600 and iproveratril. This suggests that the phasic Ca entry involves the late Ca channel.9. Recovery of the outward K current after a long depolarization is much faster than recovery of the late Ca entry system. This provides further support for the view that the late Ca channel and the K channel are distinct.

Animals↗
PubMed1972

Binding of the cardiac glycoside ouabain to intact cells.

1. Measurements were made of the binding of [(3)H]ouabain to a variety of cell types.2. Two components of binding could usually be distinguished: a component that saturated at low glycoside concentrations and a component that increased up to the highest ouabain concentrations examined.3. Detailed studies with HeLa cells and kidney slices from guinea-pigs showed that the saturable component is probably associated with inhibition of the Na pump. The main evidence for this is (a) at low concentrations of ouabain there is a close correspondence between the concentration of ouabain giving half-maximum binding and the concentration giving half-maximum inhibition of the Na pump; (b) at low glycoside concentrations, binding precedes inhibition of the Na pump; (c) the rate of binding is very sensitive to external K ions, being highest in the absence of K; (d) binding is reversible and the release of ouabain is associated with reactivation of the Na pump, (e) binding is reduced in the absence of Na ions and in the presence of metabolic inhibitors; (f) binding has a Q(10) of about 4; and (g) in the presence of Na and ATP, lysed HeLa cells bind a similar amount of ouabain and the binding is sensitive to K ions.4. The linear component of binding does not seem to involve the Na pump and it may reflect uptake of ouabain into the cell interior. It has a Q(10) of 2.5 and is unaffected by K concentrations which have a large effect on the saturable component.5. Bound ouabain could be removed from HeLa cells by low pH, trichloroacetic acid, urea, high temperatures and 100% ethanol. These agents did not distinguish between the two components of binding.6. Criteria are developed for estimating the number of Na pumping sites in cells and the data for ouabain-binding to a number of cells is compared with the activity of the (Na + K)-activated ATPase in the same tissues. Although the number of pumping sites varies from less than 1/mu(2) to 1500/mu(2) of membrane, the turnover at these sites seems to be fairly constant between 3,500 and 15,000 min(-1) at 35 degrees C.

Adenosine Triphosphatases↗
PubMed1972

Inhibition of the sodium pump in squid giant axons by cardiac glycosides: dependence on extracellular ions and metabolism.

1. The rate of inhibition of the Na pump by ouabain was examined both by direct measurement of the rate of decline of the Na efflux and by the binding of [(3)H]ouabain.2. The onset of inhibition of the Na efflux was concentration-dependent; but did not follow simple first order kinetics. The time course of inhibition was roughly exponential although in about 30% of the axons inhibition was preceded by a transient stimulation of the Na efflux.3. Inhibition of the Na efflux by both ouabain and strophanthidin was apparently irreversible.4. The onset of inhibition was slowed markedly at low temperatures.5. Replacement of external Na by choline, dextrose or potassium slowed the rate of inhibition. Li behaved like Na and inhibition was faster in K-ASW than in choline-ASW.6. The rate of inhibition of Na-Na exchange was similar to that of Na-K exchange, but ouabain failed to bind securely to fully poisoned axons.7. Two components of [(3)H]ouabain-binding could be distinguished. A linear component which probably reflects uptake into the cells and a saturable component which seems to reflect binding to Na-pumping sites.8. The saturable component of binding followed a similar time course to the inhibition of the Na efflux and the rate of binding was reduced in choline-ASW and in fully poisoned axons.9. Measurements of [(3)H]ouabain-binding indicate that the number of Na pumping sites in the axon membrane is probably between 10(3) and 10(4)/mu(2).

Animals↗
PubMed1972

Mobility and transport of magnesium in squid giant axons.

1. The Mg content of axons obtained from Loligo forbesi averaged 6.4 +/- 0.8 m-mole/kg axoplasm.2. A small patch of radioactive (28)Mg injected into an axon broadened considerably. A similar patch of (45)Ca showed hardly any broadening. The self-diffusion coefficient of Mg in axoplasm is about 2 x 10(-6) cm(2)/sec which is at least twenty times greater than that of Ca.3. Under the influence of an applied electric field Mg migrated towards the cathode. Its mobility was about half of that of Mg in free solution. This suggests that the concentration of ionized Mg in squid axoplasm is between 2 and 3 m-mole/kg axoplasm. The mobility of Mg was not changed by poisoning the axon fully.4. Mg influx and Mg efflux were roughly the same and equal to about 1 p-mole/cm(2) sec. Mg efflux was reduced by poisoning with cyanide and by replacement of external Na by choline. Removal of external K or Ca had little effect and removal of external Mg tended to increase the efflux.5. The dependence of Mg efflux on Na seems not to be secondary to changes in Ca because it persists in the absence of external Ca and in axons pre-injected with EGTA. The form of the dependence on Na ions approximates to a simple rectangular hyperbola.6. Replacement of external Na by Li or choline increased Mg influx. Mg influx was unaffected by cyanide.7. Mg efflux was reduced to an average of 15% by poisoning with cyanide or DNP. The efflux could be recovered by injection of ATP. Inhibition persisted in axons pre-injected with EGTA, showing that it is not secondary to a rise in Ca(i).8. During nervous activity there is an extra entry of Mg. For axons immersed in sea water this extra Mg entry per impulse is roughly the same as the extra Ca entry per impulse.

Action Potentials↗
PubMed1971

Depolarization and calcium entry in squid giant axons.

1. Changes in ionized calcium in giant axons were followed by recording the light produced by injected aequorin.2. From the effect of injecting calcium buffers the internal concentration of ionized calcium was found to be about the same as in a mixture of 45 Ca EGTA:55 free EGTA, i.e. about 0.3 muM.3. After an axon had been exposed to cyanide for 50-100 min the velocity of the aequorin reaction increased about 500 times. This effect, which could be reversed rapidly by removing cyanide, was probably brought about by release of calcium from an internal store.4. Injecting 30 mumole ATP per litre of axoplasm into a cyanide-poisoned axon caused a transient lowering of light intensity; oligomycin blocked the effect.5. Raising external calcium or replacing external sodium by choline or lithium reversibly increased the light produced by axons injected with aequorin.6. Stimulation at 50-200 impulses/sec in a solution containing 112 mM-Ca caused the light intensity to increase to a new steady level; after stimulation the light intensity returned to its original level with a time constant of 10-30 sec. Similar but smaller effects were seen in solutions containing less external calcium. The recovery after stimulation is probably due to uptake of calcium by the internal store.7. Injecting 3 m-mole EGTA per litre axoplasm lowered the resting glow and abolished the aequorin response to stimulation.8. There was no light response to stimulation immediately after an axial injection of aequorin and the effect increased to a ;steady' level with a half-time of about 5 min. The conclusion is that the rise in calcium concentration resulting from stimulation is confined to the peripheral part of the axon and that the diffusion coefficient of aequorin in axoplasm is about 4 x 10(-7) cm(2)/sec.9. The increment in light per impulse often increased markedly during the course of a long experiment and there was also considerable variation between axons.10. If the light response to stimulation was small it was proportional to the frequency of stimulation; if large to the square of the frequency.11. Voltage-clamp experiments showed that the calcium entry associated with a depolarizing pulse could be divided into an early component which was abolished by tetrodotoxin (TTX), and a late component which was unaffected by this inhibitor.12. The time relations of the early calcium entry were consistent with its being a leak of calcium ions through the sodium channel; the permeability of the sodium channel to calcium was about 1% of the permeability to sodium.13. The late entry of calcium was little changed by injecting enough tetraethylammonium (TEA) to block the outward potassium current; it was greatly reduced by external concentrations of manganese which had little effect on the maximum potassium conductance.14. The voltage-response curve for the late entry of calcium had a well defined maximum and was similar in shape to the curve relating calcium entry to depolarization at the presynaptic ending (Katz & Miledi, 1969, 1970).

Action Potentials↗
PubMed1971

Sodium transport by perfused giant axons of Loligo.

The sodium efflux from perfused squid giant axons has been studied using radioactive sodium, and the sufficient conditions for the maintenance of a potassium- and ouabain-sensitive sodium efflux have been established. The following were found.1. Axons extruded and then perfused with their own axoplasm had a sodium efflux which was sensitive to cyanide, potassium and ouabain and was thus similar to the efflux from intact axons.2. A method for replacing natural axoplasm into fibres previously perfused with artificial axoplasm was developed and used to establish an artificial perfusate that was not irreversibly toxic.3. Short perfusion (5 min) with a variety of artificial perfusates was then found to give fibres which had potassium- and ouabain-sensitive sodium effluxes when ATP was present in the perfusate.4. In the absence of ATP the sodium efflux was small and relatively insensitive to both external potassium and to ouabain.5. With ADP in the perfusate, fibres gave a sodium efflux which was ouabain-sensitive but was little affected by the removal of external potassium from the sodium-rich sea water bathing the fibres.6. The perfused fibres differed from intact fibres in having large ouabain-insensitive sodium effluxes.7. After very long perfusions (40-90 min), with the simple media containing ATP, the rate constant for sodium efflux from the fibres tended to be large and was relatively insensitive to potassium or to ouabain.8. Fibres refilled with natural axoplasm after long perfusion showed increased sensitivity to external potassium; refilled with dispersed axoplasm the sodium efflux tended to become very large.9. After very long perfusions with artificial axoplasms containing ATP, a potassium- and ouabain-sensitive sodium efflux was found to persist provided that dextran was present and the total osmotic pressure and the hydrostatic pressure of the perfusate were controlled. Under these conditions the sodium efflux resembled that from briefly perfused fibres. The necessary and sufficient conditions for the maintenance of sodium transport by perfused giant axons are discussed.

Adenosine Diphosphate↗