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P F Baker

Publications and source records attributed to P F Baker.

At least 55 records · Page 3Linked to original sources
PubMed1980

Anaesthetics increase light emission from aequorin at constant ionised calcium.

The isolation by Shimomura, Johnson and Saiga of a protein, aequorin, that emits light in the presence of micromolar concentrations of ionised calcium opened up new possibilities for the study of ionised calcium inside cells. It is a relatively simple matter to introduce aequorin into large cells by microinjection, and the rate of light emission gives a direct measure of intracellular free calcium. During an investigation into the action of certain anaesthetics on squid axons, we noticed that these agents always increase the light output from intracellular aequorin. Subsequent analysis has now revealed that this does not result from a rise in ionised calcium inside the axon but seems to reflect a direct effect of the anaesthetic agent on the aequorin molecule. The agents studied all produce greater activation of the light emitting reaction at a constant level of ionised calcium. These rather simple observations have several important biological implications: (1) aequorin might be an interesting model system for studying interaction between anaesthetics and proteins, (2) other Ca-sensitive proteins might behave like aequorin, their affinity for calcium being subject to modulation, and (3) such modulation of endogenous Ca-binding proteins might contribute to the mechanism of anaesthesia.

Aequorin↗
PubMed1980

Gaining access to the site of exocytosis in bovine adrenal medullary cells.

1. A new technique is described for gaining access to the intracellular site at which exocytosis is controlled. By exposing isolated cells to high electric fields of brief duration, it is possible to create "leaky" cells in which the plasma membrane has been by-passed without impairing the ability of the cells to undergo exocytosis. 2. In the presence of EGTA and an ionized Ca of less than 10(-8) M, "leaky" cells lose less than 1% of their total catecholamine. When exposed to 10 microM ionized Ca they lose up to 30% of their catecholamine together with dopamine-beta-hydroxylase but no lactate dehydrogenase. The time course of release of both catecholamine and dopamine-beta-hydroxylase are identical, suggesting that release must involve exocytosis. Exocytosis in these "leaky" cells has the following properties: A) It is half maximal in the presence of a buffered ionized Ca of approximately 1 microM. Millimolar concentrations of Mg reduce both the affinity for Ca and also the total amount of catecholamine released at high calcium concentrations. B) It has a specific requirement for Mg ATP. C) It is inhibited by a variety of anions. The most potent inhibitor is SCN, and the least glutamate and acetate. D) It is inhibited by the anti-psychotic drug trifluoperazine.

Adenosine Triphosphate↗
PubMed1979

The flow properties of axoplasm in a defined chemical environment: influence of anions and calcium.

The flow properties of axoplasm have been studied in a defined chemical environment. Axoplasm extruded from squid giant axons was introduced into porous cellulose acetate tubes of diameter roughly equal to that of the original axon. Passage of axoplasm along the tube rapidly coated the tube walls with a layer of protein. By measuring the rate of low back and forth along the tube, the rheological properties of the axoplasm plug were investigated at a range of pressures and in a variety of media. Axoplasm behaves as a classical Bingham body the motion of which can be characterized by a yield stress (theta) and a plastic viscosity (eta p). In a potassium methanesulphonate medium containing 65 nM free Ca2+, theta averaged 109 +/- 46 dyn/cm2 and eta p1 146 +/- 83 P. These values were little affected by ATP, COLCHICINE, CYTOCHOLASIN B or by replacing K by Na but were sensitive to the anion composition of the medium. The effectiveness of different anions at reducing theta and eta p1 was in the order SCN greater than I greater then Br greater than Cl greater than methanesulphonate. Theta and eta p1 were also drastically reduced by increasing the ionized Ca. This effect required millimolar amounts of Ca, was unaffected by the presence of ATP and was irreversible. It could be blocked by the protease inhibitor TLCK. E.p.r. measurements showed that within the matrix of the axoplasm gel there is a watery space that is largely unaffected by anions or calcium.

Animals↗
PubMed1978

Uptake and binding of calcium by axoplasm isolated from giant axons of Loligo and Myxicola.

1. Axoplasm isolated from giant axons of the squid Loligo and of the polychaete worm Myxicola continues to bind Ca and maintain an ionized Ca concentration close to 0.1 microgram which is similar to that seen in intact axons. 2. Injection of Ca into isolated axoplasm only produces a transient rise in ionized Ca showing that axoplasm can buffer a Ca challenge. 3. In order to characterize the Ca-binding systems isolated axoplasm was placed in small dialysis tubes and exposed to a variety of artificial axoplasms containing 45Ca. 4. In the presence of ATP, orthophosphate and succinate, Ca uptake appreciable and after 4 hr exposure of Loligo axoplasm to 0.1 microgram-Ca, approximately 100 mumole Ca/kg axoplasm was bound. Binding could be divided operationally into two distinct processes, one that requires ATP or succinate togeth with orthophosphate and is blocked by cyanide and oligomyocin, and one that is unaffected by these reagents. 5. Energy-dependent binding has a large capacity, but a rather low affinity for Ca, being half-maximal between 20 and 60 microgram-Ca. In Loligo, its properties closely parallel those of a crude mitochondrial preparation isolated from axoplasm; but there are some interesting differences in Myxicola. Energy-independent binding is half-maximal at ionized Ca concentrations between 80 and 160 nM but is readily saturated and has a capacity of 6-60 mumole/kg axoplasm. 6. Ca binding by Loligo is greatest in media containing roughly physiological concentrations of K and is reduced by isosmotic replacement of K by Na. This effect seems to be confined to the energy-dependent, presumed mitochondrial, component of binding. 7. Ca binding by Loligo axoplasm is reduced by both La and Mn ions.

Adenosine Triphosphate↗
PubMed1978

The influence of extracellular calcium binding on the calcium efflux from squid axons.

1. The Ca efflux from unpoisoned squid axons seems to consist of three components: Cao-dependent Ca efflux and a 'residual' flux that persists in the nominal absence of external Ca and Na and is little affected by the further removal of external Mg. 2. Evidence is presented to show that much of the apparent dependence on Cao of the Ca efflux from unpoisoned axons probably does not reflect a membrane process but results instead from the existence of a Ca-binding matrix external to the plasma membrane. Removal of external Ca produces a fall in efflux which is not maintained but returns to its original level over 20-60 min. Readmission of Ca produces only a transient increase in Ca efflux. Addition of EGTA to nominally Ca-free media also produces only a transient rise in Ca efflux. 3. Direct measurement of Ca binding to the surface of highly cleaned axons reveals appreciable binding over a wide range of Ca concentrations. A high affinity component of superficial binding can be recognized which has a capacity of about 60 p-mole/cm2 axon membrane and is half-maximal about 0.3 micrometer-Ca in Na-ASW. This component of binding is unaltered in cyanide-poisoned axons and in media in which Na is replaced isosmotically by Tris; but is reduced in the presence of 1 mM-La or after brief exposure of the axon to pronase. There is also a component of large capacity and lower affinity which was not saturated by 100 mM-Cao. 4. After brief pronase treatment the sensitivity of the Ca efflux to external Ca is markedly reduced although the Na-dependent Ca efflux persists apparently unaltered. 5. Addition of La produces a transient increase in Ca efflux followed by a maintained fall. 300 micrometer-La largely inhibits the component of the Ca efflux that persists in the nominal absence of external Na and Ca. Higher concentrations of La also inhibit Nao-dependent Ca efflux. 6. The Cao-dependent Ca efflux and Nao-dependent Ca efflux seen in poisoned axons persist in the presence of EGTA or after pronase treatment, suggesting that both are genuine membrane processes. 7. We conclude that in unpoisoned axons 50-90% of the Ca efflux can continue in the absence of external Ca, Na and Mg and may reflect an uncoupled extrusion of Ca. Most of the remaining efflux from these axons is Nao-dependent although a small Cao-dependent component can be distinguished in the absence of Na. Poisoning results in the loss of uncoupled Ca efflux, the appearance of appreciable Cao-dependent Ca efflux and alteration in the kinetics of Nao-dependent Ca efflux.

Animals↗