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Biomedical subjects

K E Akerman

Publications and source records attributed to K E Akerman.

At least 91 records · Page 5Linked to original sources
PubMed1989

Depolarization of human neuroblastoma cells as a result of muscarinic receptor-induced rise in cytosolic Ca2+.

The role of intracellular free Ca2+ in muscarinic-receptor linked depolarization of SH-SY5Y neuroblastoma cells has been determined by using the bisoxonol membrane potential probe DiBaC4-(3) and intracellular Ca2+ indicator fura-2 respectively. Carbachol and the Ca2+ ionophore, ionomycin, at concentrations which caused similar rises in intracellular Ca2+ increased the bisoxonol fluorescence (depolarization) to the same extent. The membrane potential responses, but not the changes in intracellular Ca2+, were dependent on extracellular Na+. Ionomycin depletion of intracellular Ca2+ with EGTA and ionomycin or loading the cells with a Ca2+ buffer, BAPTA, reduced the carbachol-induced depolarization. The results suggest that a rise in intracellular Ca2+ may cause depolarization through an increase in the Na+ permeability.

Calcium↗
PubMed1989

Glutamate receptor-linked changes in membrane potential and intracellular Ca2+ in primary rat astrocytes.

Kainate-, quisqualate- and glutamate-induced depolarization and mobilization of intracellular Ca2+ was determined in primary cultured astrocytes using the fluorescent probes DiBa-C4-(3) and fura-2, respectively. All three receptor agonists depolarized the cells in a Na+-dependent manner and increased the intracellular Ca2+ concentration. The glutamate- and quisqualate-induced increase in cytosolic Ca2+ was only partially inhibited by removal of extracellular Ca2+, whereas the response to kainate was totally dependent on extracellular Ca2+. The mechanisms for depolarization and increases in cytosolic Ca2+ appeared to be independent of each other, as extracellular Ca2+ removal or intracellular Ca2+ buffering with entrapped BAPTA did not affect the depolarization. Removal of extracellular Na+ did not affect the agonist-induced increase in Ca2+. If quisqualate was added after kainate, the cells were hyperpolarized in a Ca2+- and K+-dependent manner. This could be due to effects on a Ca2+-dependent K+ channel, the effects of which are normally hidden by the greater effect on Na+ channels as a response to quisqualate.

Animals↗
PubMed1989

Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells.

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

86Rubidium release from cultured primary astrocytes: effects of excitatory and inhibitory amino acids.

The effects of high K+, glutamate and its analogue, kainate, on K+ release were studied in primary astrocyte cultures prepared from newborn rat brains using 86Rb+ as a tracer for K+. An increase in 86Rb+ release was observed when the extracellular K+ concentration was elevated (10-40 mM). Glutamate and kainate stimulated the release in a dose-dependent manner, 100 microM concentrations being about as equally effective as high K+ (40 mM). Both compounds also caused an increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine, indicating depolarization of the membrane. No significant Na+-dependent uptake of [3H]kainate occurred in the cells, thus excluding the possibility that depolarization was due to electrogenic uptake of amino acid into the cells. GABA and taurine significantly depressed the high K+- and glutamate-induced 86Rb+ release. Taurine itself caused a small increase in 86Rb+ release and the membrane was depolarized, judging from the increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine. No effect of taurine was observed when the Cl- concentration was reduced in the experimental medium. The results suggest that cultured astrocytes respond by membrane depolarization to high external K+ and to glutamate and kainate. The degree of this depolarization can be modified by the inhibitory amino acids GABA, taurine and glycine, the effect of taurine probably being mediated by an increase in Cl- conductance across the cell membrane. The role of functional receptors for amino acid transmitters and the effects observed are discussed.

Amino Acids↗
PubMed1988

Activators of protein kinase C and phenylephrine depolarize the astrocyte membrane by reducing the K+ permeability.

The membrane potential of astrocytes has been measured by monitoring the absorbance of a cyanine dye DiS-C2-(5). Ba2+, the phorbol ester 12-tetradecanoylphorbol myristateacetate (TPA) and the diglyceride, dioctanoylglycerol (DiC8) depolarize the membrane. Valinomycin which makes the membrane potential dependent on the K+ electrochemical potential evokes a hyperpolarization when added subsequently. The alpha-adrenergic receptor agonist phenylephrine was blocked by Ba2+, TPA, DiC8 and valinomycin. The results suggest that a protein kinase C-mediated reduction in the K+ permeability is responsible for the depolarizing effect of TPA, DiC8 and phenylephrine.

Animals↗
PubMed1988

The effect of K+ and glutamate receptor agonists on the membrane potential of suspensions of primary cultures of rat astrocytes as measured with a cyanine dye, DiS-C2-(5).

The cyanine dye DiS-C2-(5) was used to investigate the effect of K+ and glutamate receptor agonists on the membrane potential of whole populations of primary rat astrocytes in suspension. Increasing the external K+ concentration from 5 to 40 mM caused a depolarization of the cells. Ba2+ blocked the response to K+, whereas 4-aminopyridine had no effect on the depolarization. The effect of added external K+ was enhanced by the addition of the neutral K+ ionophore valinomycin. This supports the view that the membrane potential of primary astrocytes is dependent of the K+ gradient, and suggests that the membrane is not ideally permeable to K+ ions. Glutamate caused a depolarization of the cells which was not affected by Ba2+. In the presence of veratridine and ouabain no effect of glutamate was seen. The cells were also depolarized by the glutamate receptor agonists quisqualate, kainate and N-methyl-D-aspartate (NMDA). The response to kainate was blocked by kynurenate, which also diminished the depolarization caused by glutamate. NMDA was effective when added after kainate. The effect of the glutamate receptor agonists tested was generally smaller than that of glutamate itself, and a prior addition of one of the agonists diminished the response to glutamate. The results obtained suggest that cyanine dyes are well suited for investigating the behavior of whole populations of cultured primary astrocytes.

Animals↗
PubMed1988

Single transient potassium channels in human neuroblastoma cells induced to differentiate in vitro.

Single channel recordings were obtained from inside-out patches of cultured human neuroblastoma cells (cell line SH-SY5Y) treated with a phorbol ester, 12-o-tetradecanoylphorbol-13-acetate (TPA) to induce differentiation. An outward current reversing near the calculated reversal potential for potassium was detected. This channel is transiently active at membrane potentials between -40 and -70 mV but with preceding hyperpolarizing pulses also at more positive potentials, up to +75 mV. The current seems to consist of two components; a slowly activating component at potentials negative to -40 mV and a fast component, more sensitive to 4-aminopyridine, seen at more positive potentials.

Cell Differentiation↗
PubMed1988

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells.

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Glucose deprivation depolarizes plasma membrane of cultured astrocytes and collapses transmembrane potassium and glutamate gradients.

Primary cultures of astrocytes were used to investigate the effects of glucose deprivation on plasma membrane potential, on the respiration and on the energy status of these cells. Plasma membrane potential, as monitored with a cyanine dye, 3,3'-diethylthiadicarbocyanine, hyperpolarized by about 100% when glucose was added to substrate-deprived cells. The effect of glucose was prevented by iodoacetate or ouabain. In the absence of glucose, cellular adenosine triphosphate/adenosine diphosphate ratio was extensively reduced and pyruvate was unable either to restore energy status or to hyperpolarize the plasma membrane of astrocytes, although it was the preferential substrate for mitochondria within the cells. Glucose deprivation and inhibition of glycolysis or respiration in the presence of glucose caused dramatic decrease in transmembrane potassium ion and L-glutamate gradients. The gradients were not restored in the presence of pyruvate. Thus, aerobic glycolysis, rather than oxidation of pyruvate, is required to maintain maximal plasma membrane potential, adenosine triphosphate/adenosine diphosphate ratios as well as K+ and L-glutamate gradients. This evidence, together with the unresponsiveness of astrocyte respiration to ouabain, indicates a functional dissociation between energy dissipation at the plasma membrane and mitochondrial synthesis of adenosine triphosphate. The results are discussed with regard to the vulnerability of glia at low levels of blood glucose and the contribution of glial dysfunction to development of hypoglycaemic encephalopathy.

Adenosine Triphosphate↗
PubMed1987

Lack of correlation between calcium mobilization and respiratory burst activation induced by chemotactic factors in rabbit polymorphonuclear leukocytes.

Low concentrations of FMLP, partially purified rabbit C5a, leukotriene B4 and platelet activating factor induced a rapid rise of intracellular free Ca2+ in rabbit polymorphonuclear leukocytes. However, the four factors differed markedly in their ability to activate the respiratory burst. The peptides FMLP and C5a induced a single, strong chemiluminescence response whereas the lipids leukotriene B4 and platelet activating factor induced a markedly less intense response with a two-peak profile. Respiratory burst activation by the peptides was dependent on extracellular Ca2+ whereas the lipids required both Mg2+ and Ca2+. The results indicate that mobilization of intracellular Ca2+ is insufficient by itself to induce respiratory burst activation and that the intracellular pathways leading to activation differ depending on the nature of the stimulus.

Animals↗
PubMed1987

Intracellular free magnesium in synaptosomes measured with entrapped eriochrome blue.

The free Mg2+ concentration within synaptosomes has been measured with an entrapped Mg2+ indicator, eriochrome blue. Ionophores gramicidin and A23187 slowly increased the absorbance of the entrapped dye. Calibration of the dye response in a Na+-based medium gave a value around 0.3 mM for the internal free Mg2+ concentration at 1 mM external Mg2+. The replacement of Na+ by choline increased this value to around 0.65 mM. Depolarisation with a high K+ concentration or depletion of intrasynaptosomal ATP with FCCP and iodoacetate did not affect the level of intracellular free Mg2+ concentration. An elevation of the external Ca2+ concentration significantly reduced internal Mg2+ to about 0.1 mM. Ca2+ had no significant effect when Na+ was replaced by choline. The results indicate that the intrasynaptosomal Mg2+ activity is partially regulated by a Na+-Mg2+ exchange mechanism which does not directly require ATP as an energy source.

Adenosine Triphosphate↗
PubMed1987

Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells.

Muscarinic receptor-linked increases in intracellular free Ca2+ as measured with quin-2 and Ca2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca2+ and stimulation of Ca2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3H-NMS, by a non-labelled agonist was studied.

Calcium↗
PubMed1987

Ionic dependence of membrane potential and glutamate receptor-linked responses in synaptoneurosomes as measured with a cyanine dye, DiS-C2-(5).

Membrane potentials of particles present in a subcellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absorbance of a cyanine dye, DiS-C2-5. The membrane potential of the particles seems to be dependent on both Cl- and K+ diffusion potentials, as judged from dependence of the absorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl- was replaced with gluconate. The apparent high Cl- permeability of the membrane preparation was reduced in the presence of picrotoxin, a finding suggesting endogenous activation of receptor-linked Cl- channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4-aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N-methyl-D-aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl- permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmitter receptor-linked membrane potential changes with optical probes of membrane potential.

Animals↗
PubMed1986

Measurement of cytoplasmic, free magnesium concentration with entrapped eriochrome blue in nerve endings isolated from the guinea pig brain.

Cytoplasmic, free Mg2+ was measured spectrophotometrically using an intrasynaptosomally entrapped Mg2+-indicator, Eriochrome blue (EB). Addition of the ionophore A23187 or disruption of the synaptosomal plasma membrane with digitonin caused an increase in absorbance of entrapped EB with a maximum at 551 nm, which is typical for the Mg2+-EB complex. A conversion of absorbance changes to levels of free Mg2+ concentrations was performed after disruption of synaptosomal plasma membranes by digitonin. The results indicated that the internal, free Mg2+ increased from 0.34 to 2.2 mM when the extracellular Mg2+ concentration was increased from 1 to 5 mM. The low values of cytoplasmic, free Mg2+ concentrations suggest the presence of effective regulatory mechanisms in the nerve endings.

Animals↗
PubMed1986

Quantitative measurements of the cytosolic Ca2+ activity within isolated guinea pig nerve-endings using entrapped arsenazo III and quin2.

The absorbance changes of intrasynaptosomally entrapped arsenazo III have been converted into values of free Ca2+ concentration by correcting for the nonlinear response of arsenazo III at different concentrations of the dye as well as for changes in internal pH. An average resting value for free Ca2+ concentration around 0.4 microM is obtained. Depolarization with veratridine or gramicidin increases this value to around 3 microM. Measurements of cytosolic free Ca2+ with the quin2 method gives much lower values in similar conditions. The release of prelabelled [14C]noradrenaline from the nerve-endings is maximally activated when the internal free Ca2+ concentration rises as measured with arsenazo III to about 4 microM when titrated with increasing concentrations of ionophore A23187.

Aminoquinolines↗
PubMed1986

Development of a neural phenotype in differentiating ganglion cell-derived human neuroblastoma cells.

Human neuroblastoma cells (clone SHSY-5Y) induced to differentiate by 12-O-tetradecanoylphorbol-13-acetate (TPA) are shown to possess properties characteristic of mature ganglion cells. Elevation of the external K+ concentration, exposure to Ca2+ ionophore A23187, and acetylcholine all stimulate the release of preloaded 3H-noradrenaline in the presence but not in the absence of added Ca2+. Acetylcholine causes a fall in the 86Rb+ or 14C-TPMP equilibrium potential across the plasma membrane and stimulates 86Rb+ efflux. These responses are prevented by atropine. Acetylcholine and muscarine but not nicotine stimulate an increase in 45Ca2+ influx, an effect blocked by atropine. None of these responses have been observed in nondifferentiating cells. Muscarinic receptors, however, as measured by the binding of tritiated quinuclidinyl benzilate (3H-QNB), were present to a similar extent in control and differentiated cells. Both cell types also exhibit an accelerated release of Ca2+ in response to acetylcholine, but the control cells were at least 1 order of magnitude more sensitive to the agonist.

Acetylcholine↗
PubMed1985

Dependence of cytoplasmic calcium transients on the membrane potential in isolated nerve endings of the guinea pig.

The relation of changes in internal, free Ca2+, measured with arsenazo III, to the membrane potential, measured with the cyanine dye di-S-C2(5) or 86Rb+ distribution ratio, was studied in isolated guinea pig cortical nerve endings. Depolarization of the plasma membrane with veratridine or gramicidin as well as addition of ionophore A23187 led to an increase in cytosolic Ca2+. Only the response to veratridine was inhibited by tetrodotoxin. The dependence of the depolarization-induced increase in intraterminal, free Ca2+ on the membrane potential between about -50 to 0 mV was sigmoidal. A maximal increase in cytosolic Ca2+ was reached when the membrane potential was depolarized from the resting level, about -64 mV, to about -40 mV. These results show that in isolated nerve endings the activation of voltage-sensitive Ca2+ channels concomitantly leads to an increase in cytosolic, free Ca2+. Comparison of the results of the present study with the previous electrophysiological observations indicate that Ca2+ channels in synaptosomes, presynaptic nerve terminals of the squid giant synapse and cardiac cells have essentially similar voltage dependency.

Animals↗