Search PubMed⌕ Search

Biomedical subjects

K E Akerman

Publications and source records attributed to K E Akerman.

At least 127 records · Page 7Linked to original sources
PubMed1982

Ca2+ transport and cell activation.

The role of the Ca2+ ion as a second messenger in various processes of cell activation is reviewed. In a resting cell the cytosolic Ca2+ activity is about 10(-7) M. When activated there is a Ca2+ inflow into cells or Ca2+ release from internal storage sites, which lead to an increase in the cytosolic Ca2+ activity to 10(-6)-10(-5) M. This subsequently leads to an activation of any Ca2+ sensitive processes within cells. Such activities include muscle contraction, secretion, energy metabolism, mitosis or meiosis. Pharmacological aspects of cellular Ca2+ metabolism are also discussed.

Animals↗
PubMed1981

Biochemical approaches to the study of cytosolic calcium regulation in nerve endings.

The nerve ending cytosol is bounded by the plasma membrane, the mitochondrial inner membrane and the endoplasmic reticulum membrane, transport across each of which is capable, in theory, of regulating the cytosolic free Ca2+ concentration. By parallel monitoring of mitochondrial and plasma membrane potentials, ATP levels, Na+ gradients and intrasynaptosomal Ca2+ distribution in preparations of isolated synaptosomes, we conclude the following: (a) mitochondria in situ represent a major Ca2+ pool, regulating the upper steady-state limit of the cytosolic free Ca2+ concentration by sequestering Ca2+ reversibly; (b) this limit is responsive to the cytosolic Na+ concentration, but is below the concentration required for significant exocytosis; (c) plasma membrane Ca2+ transport can be resolved into a constant slow influx, a voltage-dependent and verapamil-sensitive influx and an ATP-dependent efflux, while Ca2+ efflux driven by the sodium electrochemical potential cannot be detected; (d) Ca2+ regulation by intrasynaptosomal endoplasmic reticulum appears to be of minor significance in the present preparation.

Animals↗
PubMed1981

Intrasynaptosomal compartmentation of calcium during depolarization-induced calcium uptake across the plasma membrane.

The distribution of Ca2+ between mitochondrial and non-mitochondrial compartments within intact synaptosomes is investigated during the net Ca2+ uptake induced by plasma membrane depolarization. The steady-state synaptosomal Ca2+ content (5.8 +/- 0.3 nmol/mg protein) is increased by 77% by plasma depolarization induced by veratridine plus ouabain (9.7 +/- 0.6 nmol/mg protein) and by 100% by high K+ (50 mM) (11.0 +/- 0.9 nmol/mg protein). Prior abolition of the mitochondrial membrane potential, and hence inhibition of intrasynaptosomal mitochondrial Ca2+ accumulation, decreased the steady-state Ca2+ accumulation by 40% in both the control and the veratridine-ouabain depolarization, and by almost 60% in the case of high K+ depolarization. Similar values were obtained for the release of Ca2+ from synaptosomes when the mitochondrial membrane was depolarized after a steady state had been attained. Control experiments demonstrated that contaminating free mitochondria were not responsible for the altered Ca2+ accumulation. That the decrease in the Ca2+ accumulation on mitochondrial depolarization corresponds to the extent of the mitochondrial pool was confirmed by rapid synaptosomal disruption with digitonin which gave values of 2.5 +/- 0.5 nmol/mg protein, 4.4 +/- 0.9 nmol/mg protein and 6.9 nmol/mg protein for control or veratridine/ouabain- and high-[K+]-depolarized synaptosomes, respectively. The lesser contribution of intrasynaptosomal mitochondria during veratridine/ouabain-induced depolarization is proposed to be a consequence of raised cytosolic Na+ concentrations activating the mitochondrial Ca2+ efflux pathway. The results demonstrate that intrasynaptosomal mitochondria represent a metabolically responsive Ca2+ pool in situ.

Animals↗
PubMed1981

Calcium transport by intact synaptosomes. Influence of ionophore A23187 on plasma-membrane potential, plasma-membrane calcium transport, mitochondrial membrane potential, respiration, cytosolic free-calcium concentration and noradrenaline release.

Ionophore A23187, a Ca2+/2H+ exchanger, has multiple effects on the function of isolated nerve endings (synaptosomes). (a) There is a net uptake of Ca2+, the rate of which increases with ionophore concentration. (b) When Ca2+ uptake is terminated by EGTA, the ionophore induces an apparent efflux of Ca2+. (c) The plasma-membrane potential, calculated from the 86Rb+ distribution, decreases slowly upon addition of the ionophore. (d) Release of [3H]noradrenaline induced by the ionophore consists of a rapid phase followed by a subsequent slower phase, which coincides with the fall in plasma-membrane potential. (e) Ouabain depolarizes the plasma membrane without inducing the rapid phase of transmitter release. (f) The membrane potential of intrasynaptosomal mitochondria determined in situ falls rapidly upon addition of A23187, and there is a synchronous increase in synaptosomal respiration. (g) All these effects require external Ca2+ and the same range of ionophore concentrations. (h) Using the respiratory stimulation induced by the ionophore in isolated mitochondria to calibrate the cytosolic concentration of free Ca2+ in intact synaptosomes it is concluded that the cytosolic Ca2+ required for release of [3H]noradrenaline is in the range 1-10 microM.

Animals↗
PubMed1981

Electron-dense precipitates in glomus cells of rat carotid body after fixation in glutaraldehyde and pyroantimonate-osmium tetroxide mixture as possible indicators of calcium localization.

An attempt was made to study the subcellular localization of calcium in carotid body glomus cells of adult rats using fixation with glutaraldehyde followed by treatment with a mixture of pyroantimonate and osmium tetroxide. Precipitates were seen as electron-dense particles (EDP) in the glomus cells, mostly within membrane-bound organelles, such as dense-cored vesicles, mitochondria, small clear vesicles, multivesicular bodies, and especially in lysosomes. However, EDP were also seen in the nuclei and in the free cytoplasm of the glomus cells and even outside them. Preincubation of carotid bodies in media containing calcium and either high potassium or calcium-ionophore A 23187 resulted in a marked increase in the general precipitation pattern, there being an increased amount of EDP both in the glomus cell nuclei and in the cytoplasm. Dense-cored vesicles more often showed precipitates than those in the controls. Some dense-cored vesicles contained multiple precipitates, typically located in the electron-lucent area between core and vesicle membrane. Extensive diffusion of ions probably occurred during fixation before precipitation, making the localization of calcium and other precipitating cations unreliable. However, it is possible that precipitates, which were regularly seen in the dense-cored vesicles, may reflect the content of bound calcium. The possible significance of calcium in glomus cell function is discussed, and the need for more adequate methods is emphasized.

Animals↗
PubMed1981

Inhibition and stimulation of respiration-linked Mg2+ efflux in rat heart mitochondria.

Respiration-driven Mg2+ efflux from rat heart mitochondria has been studied in different conditions. Almost total release of Mg2+ from the mitochondria occurs upon addition of a proton/bivalent cation exchanger, A23187. The content of Mg2+ remaining in mitochondria after A23187 treatment is the same if part of the mitochondrial Mg2+ has already been extruded through the energy-linked mechanism. Some inhibition of Mg2+ efflux is observed in the presence of high concentrations of La3+ (100 micro M). A proton/monovalent cation exchanger, nigericin, completely prevents Mg2+ efflux, whereas a cation conductor, valinomycin, considerably stimulates it. The results indicate that the main part of mitochondrial Mg2+ is present in the membrane-bounded compartment, probably in the matrix space. The driving force of the Mg2+ efflux appears to be the proton gradient (deltapH) created by mitochondrial respiration.

Animals↗
PubMed1981

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange.

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.

Animals↗
PubMed1980

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

The association of Ca2+ with isolated nerve endings (synaptosomes) is investigated and resolved into two components, that bound to the outer surface of the plasma membrane and that transported across the plasma membrane. When synaptosomes are added directly to a Ca2+-containing medium, there is an initial rapid uptake of Ca2+ across the plasma membrane, followed by a slow uptake that proceeds for 20 min. The rapid phase is not observed if the synaptosomes are initially pre-incubated in a Ca2+-free medium. Rapid disruption of synaptosomes reveals that less than 3 nmol of transported Ca2+ per mg of synaptosomal protein can be ascribed to non-mitochondrial components, whereas the remainder, up to 79% of the total, is further transported into the mitochondrial matrix. Abolition of oxidative phosphorylation while the mitochondrial membrane potential is retained leads to a time-dependent increase in transported Ca2+, whereas abolition of the mitochondrial membrane potential decreases both plasma-membrane transport and accumulation of Ca2+ in the mitochondrial matrix. It is concluded that intrasynaptosomal mitochondria are major regulators of synaptosomal Ca2+.

Animals↗
PubMed1980

Effects of ionophores and metabolic inhibitors on the mitochondrial membrane potential within isolated hepatocytes as measured with the safranine method.

A difference spectrum with a peak of absorbance at 526nm appears slowly upon addition of valinomycin or KCN in combination with oligomycin to a hepatocyte suspension in the presence of safranine. When the cells are incubated at 37 degrees C in a medium containing safranine, a slow decrease in the absorbance occurs at the wavelength pair 524-484 nm. The change in absorbance is completed within 20-30 min after additions of cells to a medium containing safranine. At this time the safranine concentration of the outer medium is considerably decreased. The safranine signal is completely reversed by valinomycin, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or KCN in combination with oligomycin. None of these treatments have any immediate effect on cellular ATP concentrations or the 36Cl- equilibrium potential across the plasma membrane. In the presence of iodoacetate a slow reversal of the trace can be induced upon addition of KCN, but not of oligomycin alone. Rotenone, in combination with oligomycin, does not reverse the safranine signal except when both KF and iodoacetate are present, in which case a slow reversal is seen. A subsequent addition of duroquinone brings back the signal to the same level as in the presence of rotenone alone. The results indicate that the spectral response of safranine in the presence of isolated hepatocytes is a result of a slow penetration of safranine into intracellular mitochondria, where aggregation of safranine molecules occurs as a response to the mitochondrial membrane potential.

Animals↗
PubMed1980

Measurements of membrane potentials in Escherichia coli K-12 inner membrane vesicles with the safranine method.

The use of safranine, a positively-charged dye, as a probe for the determination of membrane potentials in Escherichia coli vesicles has been studied. 1. Shifts in the spectrum of safranine were observed during induction of potassium ion diffusion potentials with valinomycin or during oxidation of formate by vesicles prepared from cells of E. coli K-12 or ML 308-225 subjected to anaerobic growth with nitrate. The extent of the valinomycin-dependent spectral change correlated linearly with the magnitude of the K+ equilibrium potential, as calculated from the Nernst equation, from 50 to 160 mV (interior negative). The formate-induced changes could also be calibrated by increasing the concentration of potassium in the presence of valinomycin, after the formation of formate-dependent responses. In this case, results identical to those obtained with the first method were obtained. 2. O2 or nitrate-dependent oxidation of formate resulted in a membrane potential of the order of 170 mV. The oxidation of ascorbate-reduced N-methylphenazonium methosulphate resulted in a potential of similar magnitude, but anaerobically with nitrate only a small but definite potential was formed. 3. The water-soluble quinones, duroquinone and menadione, could produce membrane potentials when used in their oxidized or reduced forms in the presence of formate or nitrate (or oxygen). 2-Hydroxy-1,4-naphthoquinone was not only ineffective but was found to be inhibitory. 4. N,N'-dicyclohexylcarbodiimide at suitable concentrations increased the rate of formation and the extent of membrane potentials induced by respiration or by artificial means.

Biological Transport↗
PubMed1980

Thermodynamic aspects of translocation of reducing equivalents by mitochondria.

The "poise" in which there is no net transport of reducing equivalents mediated by glutamate and aspartate movements across the mitochondrial membrane has been studied using nonrespiring liver mitochondria, supplied with a given extramitochondrial ATP/ADP ratio. Absorbance shifts in safranine have been used as a probe to estimate the membrane potential generated under these conditions. Measurements of the asymmetric distribution of substrate anions and of reducing potential across the limiting membrane gave estimated differences of between 2.2 and 3.3 kcal when the ATP/ADP ratio in the suspending medium was about 30. The estimated membrane potential under these conditions was about 140 mV. All of the above parameters were decreased in a corresponding manner when the ATP/ADP ratio imposed from outside was decreased. A strong inflection in the slope of correlated parameters was obtained when the ATP/ADP ratio was below about 0.5. On the other hand, there was a linear relationship between the membrane potential and the asymmetric poise of reducing equivalents and of carbon metabolites. It is concluded that the poise of reducing equivalents mediated by the malate-aspartate shuttle is determined principally by the membrane potential through its effect on the electrogenic exchange of glutamate and aspartate.

Adenosine Diphosphate↗