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P F Baker

Publications and source records attributed to P F Baker.

At least 37 records · Page 2Linked to original sources
PubMed1985

The chromaffin granule proton pump and calcium-dependent exocytosis in bovine adrenal medullary cells.

Calcium-dependent exocytosis in 'leaky' bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in 'leaky' cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measured in situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.

Adenosine Triphosphate↗
PubMed1985

Calcium clamp of the intracellular environment.

Quantitative analysis of the effects of calcium on cell function requires methods for altering intracellular free Ca in a precise and reproducible manner. Microinjection of Ca is very unreliable largely because of the powerful Ca-binding properties of cytoplasm. Much more satisfactory are microinjection of Ca-buffers - provided enough buffer is introduced - and various forms of intracellular dialysis and perfusion which permit full equilibration of the cell interior with a defined artificial intracellular environment.

Adrenal Medulla↗
PubMed1984

Tyrosine hydroxylase in "leaky" adrenal medullary cells: evidence for in situ phosphorylation by separate Ca2+ and cyclic AMP-dependent systems.

The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the other on cyclic AMP. The Ca2+-dependent system is half-maximally activated by 1-2 microM Ca2+ and 0.5 mM ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 mM) does not inhibit significantly Ca2+-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 microM cyclic AMP and about 0.3 mM ATP. Ca2+-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10(-8)M Ca2+ plus 2 X 10(-5)M cyclic AMP or of 10(-5)M Ca2+, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca2+-dependent system, whereas the other site(s) are phosphorylated by both the Ca2+- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.

Adrenal Medulla↗
PubMed1983

Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function.

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 microM), a drug that inhibits the action of the calcium-dependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin, N-ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.

Actins↗
PubMed1983

Stimulus-secretion coupling in isolated bovine adrenal medullary cells.

Adrenal medullary cells can be obtained in high yield by protease digestion of slices of bovine adrenal medulla. The properties of catecholamine release from these isolated cells have been determined and compared with the intact perfused gland. Exposure to carbamylcholine, veratridine or high K evokes a transient increase in the rate of catecholamine release in association with dopamine beta hydroxylase but not lactate dehydrogenase. Exposure to A23187 releases catecholamine together with lactate dehydrogenase. In all cases release requires extracellular Ca. A comparison is made of the sensitivities of catecholamine release evoked by various agonists and the effects of nicotinic antagonists, D 600, Na-free media, TTX, Mg and trifluoperazine. Resting and carbamylcholine-evoked catecholamine release is relatively insensitive to the nature of the major extracellular anion and to the external pH over the range 6.4 to 7.8. Measurements with the dye 3 3'-Diethylthiadicarbocyanine iodide (di S-C2-(5)) show that release evoked by carbamylcholine or low concentrations of veratridine is associated--on average--with a small steady depolarization of the cells. Catecholamine release in response to nicotinic agonists, but not the other secretagogues, is greater at 20 degrees C than at 37 degrees C, possibly because of a slower rate of receptor desensitization at this temperature. Transmission electron microscopy of stimulated cells reveals a marked increase in the proportion of large membrane bounded vesicles that lack electron-dense cores. After stimulating secretion in the presence of horseradish peroxidase, the vesicles contain reaction product suggesting that they are generated by endocytosis.

Acetylcholine↗
PubMed1983

Insulin regulation of sugar transport in giant muscle fibres of the barnacle.

1. Sugar transport in the giant muscle cells of Balanus nubilus is accelerated during contractile activity and exposure to porcine insulin. The characteristics of hexose-transfer regulation in the giant muscle cells have been examined by studying the transport of 3-O-methylglucose (a non-metabolized sugar) in both intact giant fibres and fibres subjected to internal solute control by internal dialysis.2. Sugar transport in barnacle muscle is mediated by a saturable process which is inhibited by both phloretin and cytochalasin B. Insulin increases the capacity of the transport system with little effect on its apparent affinity for sugar. Under the same conditions insulin increases 3-O-methylglucose-displaceable cytochalasin B binding. The effects of insulin on transport are half-maximal at 5 muM-insulin and are abolished by both insulin antibody and phloretin. The intact barnacle releases an insulin-like material in response to a rise in blood glucose levels.3. Insulin increases the cyclic GMP (cGMP) content and reduces the cyclic AMP (cAMP) content of barnacle muscle. Experiments with fibres injected with aequorin show that insulin also lowers cytosolic ionized Ca levels. The changes in cyclic nucleotide levels induced by insulin precede the effects on sugar transport and cytosolic ionized Ca. During repetitive contractile activity, cAMP, cGMP and ionized Ca levels are raised.4. Agents which raise the cAMP content of barnacle muscle normally inhibit sugar transport. Dibutyryl cAMP also inhibits transport. Alterations in cytosolic ionized Ca levels in intact fibres are without effect on sugar transport. Nevertheless, stimulation of transport by insulin is blunted when cytosolic ionized Ca is lowered by intracellular injection of the Ca-chelating agent, EGTA.5. Sugar uptake in the internally dialysed fibre is inhibited by intracellular application of cAMP. Internal application of Ca and cGMP stimulate sugar uptake in the dialysed fibre. Cyclic AMP reduces the capacity of the transport system whereas Ca and cGMP increase the capacity of the saturable transfer system. Cyclic AMP and cGMP act at kinetically independent sites. Internal ATP (2 mM) inhibits sugar uptake in the dialysed fibre by some 40%, possibly through the production of cAMP.6. External insulin stimulates sugar uptake in the dialysed fibre even when ionized Ca levels are buffered using EGTA. Stimulation by insulin requires the presence of cytosolic ATP and is potentiated by internal application of 1 mM-GTP. In the dialysed fibre stimulation of transport by insulin is greater than that brought about by Ca and cGMP.7. The stimulation of transport by insulin in the intact fibre and its inhibition by dibutyryl cAMP are abolished by intracellular injection of Gpp(NH)p. Injection of intact fibres with GTPgammaS potentiates the stimulation of transport by insulin and renders insulin-activation of transport irreversible. Injection of intact fibres with ATPgammaS leads to the irreversible inhibition of transport.8. Injection of intact fibres with cAMP phosphodiesterase lowers cAMP levels close to zero and stimulates sugar transport. Application of insulin to diesterase-injected fibres still stimulates transport in the absence of altered cytosolic cAMP.

3-O-Methylglucose↗
PubMed1982

Metabolism and transport of strontium in giant axons of Loligo.

1. The strontium content of squid axons averages 2.9 mumol kg-1 axoplasm and squid blood contains 75 mumol Sr l-1. The bulk of the Sr inside axons is bound, possibly within mitochondria. Sr seems to be handled by squid axons in an essentially similar manner to Ca. 2. Sr uptake is increased by electrical activity, by lowering external Na, by depolarization with K and by poisoning with cyanide or FCCP. Uptake is unaffected by ouabain. 3. Sr efflux has a Q10 of 2.2. Efflux can be divided operationally into three components: one dependent on external Ca (or Sr), one dependent on external Na and a residual component that can be inhibited by external lanthanum or internal vanadate. 4. Poisoning an axon with cyanide or FCCP results in a large increase in Sr efflux which can be divided operationally into two components: one dependent on external Ca (or Sr) and one dependent on external Na.

Animals↗
PubMed1981

Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells.

Experimental analysis of the mechanisms of exocytosis and endocytosis has hitherto been hampered by the inaccessibility of the intracellular sites at which they are controlled. We have recently developed a technique that overcomes this problem. Cells are subjected to intense electric fields of brief duration; this renders the plasma membrane permeable without impairing its ability to participate in exocytosis and endocytosis. Working with 'leaky' bovine adrenal medullary cells, catecholamine release has a rather specific requirement for Mg-ATP, is activated by micromolar concentrations of ionized Ca and can be inhibited by Mg, detergents, trifluoperazine, high osmotic pressure and various anions. The mechanism of activation by Ca is discussed in some detail.

Adenosine Triphosphate↗
PubMed1981

Sugar transport in giant axons of Loligo.

1. The transport of glucose and a number of other sugars has been investigated in the giant axons of Loligo forbesi. 2. Glucose and 2-deoxy-D-glucose are phosphorylated by squid axons, alpha-methyl-D-glucopyranoside and 3-O-methylglucose are not metabolized. All four sugars can diffuse freely in axoplasm. 3. Sugar uptake in squid axons is a passive, saturable process. The maximum rate of sugar uptake increases in the order 3-O-methylglucose less than 2-deoxy-D-glucose less than D-glucose. Competition between these sugars suggests a common uptake mechanism. 4. The uptake of D-glucose but not 3-O-methylglucose or 2-deoxy-D-glucose is reduced when the external Na concentration is lowered. 5. Glucose uptake is sensitive to temperature with a Q10 for saturated uptake of 1.9 between 14.5 and 5 degrees C. Uptake is unaffected by external pH in the range 5-10 but is reduced by cyanide (2 mM). 6. Glucose and 2-deoxy-D-glucose uptake and metabolism are increased by electrical stimulation. These effects are prevented by ouabain. The uptake of 3-O-methylglucose is unaffected by stimulation. 7. The maximum rate of 3-O-methylglucose efflux is higher than the maximum rate of uptake of the sugar suggesting that transport of this sugar is asymmetric. 8. 3-O-methylglucose efflux is reduced by external sugars with order of potency 3-O-methylglucose greater than 2-deoxy-D-glucose greater than D-glucose. These effects persist when the internal 3-O-methylglucose concentration is as high as 50 mM. 9. 3-O-methylglucose efflux is inhibited reversibly by cytochalasin B and phloridzin but irreversibly by phloretin. Efflux is reduced reversibly by cyanide (2 mM). 10. 3-O-methylglucose efflux is sensitive to temperature with a Q10 of 3.2 over the range 10-20 degrees C. Efflux is unaffected by external pH in the range 6-9 but is reduced reversibly by internal acidification.

Animals↗
PubMed1981

3-O-methylglucose transport in internally dialysed giant axons of Loligo.

1. The transport of the non-metabolized sugar, 3-O-methylglucose, has been studied in the squid axon under conditions where the intracellular environment of the axon is controlled by internal dialysis. 2. Sugar transport is passive, shows saturation kinetics and is asymmetric. At 15 degrees C, the Michaelis and velocity constants for exit are approximately four times those for uptake. The asymmetry of transport is increased by raising the temperature. 3. Sugar uptake is not affected by intracellular sugar levels as high as 100 mM. Sugar exit is, however, reduced by external sugars although the apparent Km for exit is unaffected. 4. The kinetics of sugar exit under exchange conditions are determined by the kinetics of sugar uptake. These results can be accounted for by the asymmetric mobile-carrier and simultaneous-carrier models for transport. 5. Both sugar uptake and exit are reduced in the absence of ATPi. Kinetic analysis of transport under these conditions show that the capacity of the system to transport sugar is unchanged but that the affinity of the system for sugar is reduced. Internal cyclic AMP, AMP, ADP or GTP (2 mM) do not mimic this action of ATP. The hydrolysable analogue of ATP, alpha, beta-methylene-5-ATP (2 mM), (but not the nonhydrolysable analogue beta, gamma-methylene-5-ATP, 2 mM) has an ATP-like action on sugar transport. 6. Transport is unaffected by internal Ca2+ concentrations in the range 4 X 10(-8)--9 X 10(-7) M.

3-O-Methylglucose↗
PubMed1980

Insulin stimulates sugar transport in giant muscle fibres of the barnacle.

Insulin stimulates sugar transport in vertebrate skeletal muscle but the mechanism of insulin action is unknown. It has been reported that Na transport in giant muscle fibers of the barnacle (Balanus nubilis) is sensitive to insulin but no one has examined the sensitivity of sugar tansport to insulin in this preparation. We show here that insulin does, indeed, stimulate sugar transport in barnacle muscle. The great advantage of barnacle muscle over all other muscles used so far for investigating the mechanism of insulin action is its large size, which facilitates measurements on single cells and permits the experimenter to control the intracellular environment of the muscle fibre by the technique of internal dialysis. Using single muscle fibres it is possible to show that acceleration of sugar transport by insulin is associated with a fall in ionized Ca, a fall in cyclic AMP and a rise in cyclic GMP. Working with internally dialysed muscle fibres we find that insulin only increases sugar transport when the dialysis solution contains ATP. In the absence of insulin, sugar transport is dialysed muscle is increased by a rise in ionized Ca, a fall in cyclic AMP and, when the internal Ca is elevated, by a rise in cyclic GMP.

Adenosine Triphosphate↗