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Biomedical subjects

K E Akerman

Publications and source records attributed to K E Akerman.

At least 73 records · Page 4Linked to original sources
PubMed1991

High-density lipoproteins induce a rapid and transient release of Ca2+ in cultured fibroblasts.

Several different cell types showed increased rates of proliferation and cholesterol mobilization in response to treatment with high-density lipoprotein (HDL). This would suggest that one main function of HDL is the activation of signal pathways in cells. In the current study we have used the fluorescent indicator fura-2 to monitor the level of cytosolic Ca2+ ([Ca2+]i) in human skin fibroblasts. Exposure of subconfluent as well as confluent fibroblasts to HDL3 (20-60 micrograms/ml) resulted in a rapid and transient increase in [Ca2+]i. Sequential additions of HDL3 resulted in diminished rises in [Ca2+]i. The transient rise in [Ca2+]i was observed with HDL prepared from plasma either by conventional ultracentrifugation or by precipitation with dextran sulphate. Chelation of the extracellular Ca2+ with EGTA prior to the addition of HDL3 did not prevent the HDL3-induced rise in [Ca2+]i, suggesting that the mobilized Ca2+ was derived mainly from intracellular stores. Covalent modification of the apoproteins of HDL3 with dimethyl suberimidate or tetranitromethane did not inhibit the HDL3-induced rise in [Ca2+]i. This indicates that the binding of HDL3 to cell surface receptors may not be necessary for the mobilization of intracellular Ca2+. Moreover, the Ca(2+)-releasing effect of HDL3 was not inhibited by the presence of albumin (1%, w/v) in the extracellular medium, suggesting that non-esterified fatty acids were not the cause of the increased [Ca2+]i. The exposure of fibroblasts to lysophosphatidic acid, a potent mitogen and Ca(2+)-releasing agent, before addition of HDL3 completely inhibited the HDL3-induced rise in [Ca2+]i. Furthermore, phorbol 12-myristate 13-acetate blocked the HDL3-induced rise in [Ca2+]i. The results of this study imply that exposure of cells to HDL generates an intracellular signal which is induced by a component of the lipid fraction.

Albumins↗
PubMed1991

Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells.

Muscarinic receptor-linked Ca2+ mobilization and changes in cyclic AMP were studied in SH-SY5Y and IMR 32 human neuroblastoma cell lines. Muscarinic agonists acetylcholine, carbachol, methacholine and muscarine induced an increase in cytosolic free Ca2+ in a pertussis toxin (100 ng/ml)-insensitive manner in both cell lines. The ED50 values in IMR 32 cells (8-98 microM) were one order of magnitude higher than in SH-SY5Y cells (0.3-1.6 microM). Oxotremorine and pilocarpine failed to mobilize Ca2+ in IMR 32 cells. Pirenzepine antagonized carbachol-induced Ca2+ mobilization in SH-SY5Y cells with a Ki value in the range of 150-189 nM whereas the corresponding values in IMR 32 cells were 24-28 nM. Atropine inhibited a carbachol-stimulated increase in cytosolic Ca2+ with an equal potency in both cell lines (Ki 2-3 nM). Carbachol stimulated cyclic AMP (cAMP) accumulation in SH-SY5Y cells in a pertussis toxin-insensitive manner. In IMR 32 cells carbachol inhibited prostaglandin E1-stimulated cAMP accumulation. Treatment of IMR 32 cells with pertussis toxin abolished the inhibition of stimulated cAMP accumulation. These results suggest that in SH-SY5Y cells the M3 muscarinic receptor couples to both Ca2+ mobilization and stimulation of cAMP accumulation. In IMR 32 cells the M1 receptor seems to couple to Ca2+ mobilization whereas the inhibition of stimulated cAMP accumulation is coupled to a non-M1 subtype by an inhibitory G-protein.

Acetylcholine↗
PubMed1991

Effect of ethanol on gamma-aminobutyric acid and glycine receptor-coupled Cl- fluxes in rat brain synaptoneurosomes.

Chloride fluxes in synaptoneurosomes in response to additions of gamma-aminobutyric acid, glycine, and ethanol were measured using a chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). The Cl- gradient was directed outward by bathing cells in a medium low in Cl- concentration. The synaptoneurosomes responded to both gamma-aminobutyric acid and glycine by outflow of Cl- ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of Cl- ions from the synaptoneurosomes. Both picrotoxin and strychnine inhibited this effect. When the antagonists were used together, the inhibiting effect was additive. These results indicate that ethanol affects both gamma-aminobutyric acid and glycine receptor-linked chloride fluxes in the rat brain.

Animals↗
PubMed1991

Interactions of glutamate receptor agonists coupled to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture.

Changes in cytosolic free Ca2+ concentrations in response to glutamate receptor agonists and their interactions were studied in rat cerebellar granule cells grown on coverslips. The intracellular Ca2+ as measured with fura-2 increased by applying kainate (KA), quisqualate (QU), and N-methyl-D-aspartate (NMDA). The effect of KA could not be blocked by the NMDA receptor blocker 2-amino-5-phosphonovaleric acid (AP5). The KA- and QU-induced increase in intracellular free Ca2+ was also observed in a Na(+)-free medium, indicating that this response is not secondarily due to the depolarization. The effect of 10 microM QU on the KA-induced changes in cytosolic free Ca2+ was additive only at low KA concentrations, but QU at 0.1 mM totally blocked the response to KA. In the presence of 10 microM KA, the dose-response curve of QU became biphasic, whereas with 50 microM KA, a reduction of the response was seen around 1-100 microM QU. The effect of NMDA on the QU-induced response was additive only at low QU concentrations. It is proposed that rat cerebellar granule cells in primary culture express separate receptor-channel complexes for NMDA, QU, and KA, but interactions between agonists for these receptor sites exist. Thus, QU when present at intermediate concentrations seems to interact with the KA type of receptor, causing its desensitization. At high QU concentrations, an interaction of QU with the NMDA receptor site is apparent.

2-Amino-5-phosphonovalerate↗
PubMed1991

Early signal transduction in measles virus-infected lymphocytes is unaltered, but second messengers activate virus replication.

In order to understand measles virus-lymphocyte interactions, we have started to analyze factors and events which regulate measles virus infection in peripheral blood mononuclear cells (PBMC). We analyzed the initiation of cell proliferation, induced by phytohemagglutinin, in infected and control PBMC by measuring intracellular free Ca2+ by using fura-2. Measles virus-infected and control PBMC responded similarly with an increase in the amount of cytosolic free Ca2+, indicating that the early activation events are not affected and are not involved in immunosuppression. The activation signals, Ca2+ and protein kinase C, induced specifically and independently by Ca ionophore A23187 or 12-O-tetradecanoylphorbol-13-acetate (TPA), changed the restricted measles virus infection to a productive one. The combination of TPA and A23187 was the most potent activator of measles virus replication. TPA and A23187 operate through different activation mechanisms, and it is evident that measles virus replication depends on the activation of cellular signal pathways. Depletion of adherent cells enhanced virus replication, especially at the early stage of infection, indicating the inhibitory role of monocytes. Monocytes were strongly infected, but they supported complete measles virus replication only at a very low level, and virus replication could not be enhanced with TPA and/or A23187.

Adult↗
PubMed1990

Effects of bryostatins 1 and 2 on morphological and functional differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate to mature ganglion cells when treated with the phorbol ester tetradecanoylphorbol acetate (TPA). Bryostatins are a new class of protein kinase C activators that are structurally unrelated to phorbol esters. This paper describes the effects of bryostatins 1 and 2 on morphological and functional differentiation of SH-SY5Y cells. Both bryostatins induced a rapid translocation of protein kinase C from the cytosol to the membrane fraction. Within 24 h, the bryostatins had caused a nearly complete down-regulation of the enzyme. Bryostatin 1 competed for [3H]phorbol-12,13-dibutyrate binding in intact cells with potency equal to that of TPA, in contrast to bryostatin 2, which exhibited a Ki value 1 order of magnitude higher than those of the two other agents. Bryostatins induced morphological changes similar to those induced by TPA. These changes were, however, only transient, occurring during the first 6 h of incubation in the presence of these compounds. By 72 h, the cells had acquired a morphology typical of untreated cells and, although a wide range of bryostatin concentrations were used, morphological changes characteristic of differentiated SH-SY5Y cells were not detected at 72 h. Bryostatin 1 at 5 nM and bryostatin 2 at 100 nM inhibited DNA synthesis, as measured by incorporation of [3H]thymidine by SH-SY5Y cells, although to a significantly lesser degree than TPA. In spite of the fact that bryostatins failed to induce morphological differentiation in SH-SY5Y cells, these compounds down-regulated c-myc mRNA expression. Bryostatins were significantly weaker in stimulating noradrenaline synthesis, compared with TPA, and high concentrations of these agents blocked the effect of the phorbol ester when they were included together with TPA. When SH-SY5Y cells were incubated in the presence of high concentrations of bryostatins, a decreased sensitivity of cells to muscarinic agonist-induced increases in cytosolic free Ca2+ was observed. The results suggest that down-regulation of protein kinase C activity and c-myc mRNA expression do not necessarily correlate with the morphological differentiation of SH-SY5Y cells.

Antineoplastic Agents↗
PubMed1990

Coupling of glutamatergic receptors to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture.

Changes in cytosolic free Ca2+ concentrations, [Ca2+]i, in response to glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The intracellular Ca2+ as measured with fura-2 increased by applying kainate, N-methyl-D-aspartate (NMDA), quisqualate, and (RS)-d-amino-3-hydroxy-5-methyl-4-isoxazole-propionic (AMPA). When the extracellular Mg2+ was removed, the effects of NMDA and the NMDA receptor agonist cis-(+-)-1-amino-1,3-cyclopentanedicarboxylic acid (cis-ACPD) on intracellular Ca2+ were augmented. Glycine potentiated the effects of NMDA and cis-ACPD if the membrane was depolarized by increasing the extracellular K+ concentration. The NMDA receptor antagonist DL-2-amino-5-phosphonopentanic acid (AP5) abolished and the antagonist 3-([+-]-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) greatly reduced the effect of NMDA in both the normal and the Mg-free media. The dose-response curves of NMDA and, to a lesser extent, of kainate were shifted to the left, and that of quisqualate became biphasic in the Mg-free medium. The increase in [Ca2+]i produced by high quisqualate concentrations in the Mg-free medium was totally abolished by AP5. The results suggest that Ca2+ influx in cerebellar granule cells occurs through both NMDA- and non-NMDA-coupled ion channels. A part of the quisqualate-induced rise in cytosolic Ca2+ seems to be linked to the activation of NMDA receptors.

Amino Acids↗
PubMed1990

Efflux of 45calcium from cultured primary astrocytes: effects of glutamate receptor agonists and antagonists.

The effects of high-K, glutamate and glutamate receptor agonists on the efflux of Ca2+ were studied in cultured primary astrocytes, prepared from the brains of newborn rats. An increase in efflux of 45Ca2+ of produced by a large extracellular concentration of K+ was effectively inhibited by verapamil, a blocker of voltage-gated Ca2+ channels, suggesting that these cells have functional voltage-sensitive Ca2+ channels. Glutamate and its agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA) stimulated the efflux of preloaded 45Ca2+, in a dose-dependent manner. The most effective agonist was quisqualate followed by glutamate, whereas kainate and NMDA were less potent. In the Mg-free medium, the response to NMDA was significantly increased. The quisqualate receptor agonist (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also stimulated the efflux, about equally effectively as quisqualate. The glutamate-, quisqualate- and kainate-induced efflux of 45Ca2+ was significantly inhibited by L-glutamatediethylester (GDEE) and that of NMDA by DL-2-amino-5-phosphonopentanoic acid (AP5). The kainate-induced efflux was totally inhibited by verapamil, but that of glutamate only partially. No effect of verapamil was observed on the quisqualate-induced efflux of 45Ca2+. The results imply that, in cultured astrocytes, Ca2+ fluxes induced by glutamate agonists, occur partially through voltage-dependent Ca2+ channels. The extensive release of 45Ca2+ caused by quisqualate was mainly due to a release from internal stores.

Animals↗
PubMed1990

Low affinity binding to glutamate receptor sites correlates with depolarizing responses induced by glutamate and quisqualate in striatal synaptoneurosomes.

In the present study, binding affinity of glutamate and quisqualate to striatal synaptoneurosome membranes in the guinea-pig was compared with concentration-dependence of depolarizing responses induced by these agents. The displacement of radioactive glutamate from receptor binding site by unlabelled glutamate and quisqualate revealed a nonhomogeneous population of binding sites. A high affinity component of binding was observed with an inhibition constant of 0.04 microM for glutamate and 0.45 microM for quisqualate, as well as a low affinity component with an inhibition constant of 10 microM for glutamate and 87 microM for quisqualate. Changes of the membrane potential in striatal synaptoneurosomes induced by glutamate and quisqualate were detected by measuring the absorbance of a potential sensitive cyanine dye. Glutamate and quisqualate induced constantly a depolarization in synaptoneurosome particles. Concentration-response curves showed that half-maximal depolarization was obtained with 10 microM glutamate and 100 microM quisqualate. The comparison of the displacement data with the changes in the membrane potential in the present investigation indicate that in vitro glutamate and quisqualate depolarize striatal synaptoneurosome particles through low affinity binding to receptor site for glutamate.

Animals↗
PubMed1990

12-O-tetradecanoylphorbol 13-acetate and forskolin modify muscarinic receptor-linked Ca2+ mobilization in SH-SY5Y neuroblastoma cells through different mechanisms.

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.

Benzofurans↗
PubMed1989

Alpha-receptor and cholinergic receptor-linked changes in cytosolic Ca2+ and membrane potential in primary rat astrocytes.

Both phenylephrine and carbachol caused a sustained increase in Ca2+ influx and intracellular free Ca2+ of primary astrocytes as measured with 45Ca2+ and fura-2. The responses to phenylephrine and carbachol were additive, suggesting that they use different releasable pools of Ca2+. If extracellular Ca2+ was removed by EGTA only a transient rise in cytosolic Ca2+ was seen upon application of the agonists. Both compounds caused depolarization of the astrocyte membrane as determined with the optical probe 3,3-diethylthiadicarboxyamineiodide. Activation of protein kinase C with 12-tetradecanoylphorbol myristate acetate (TPA) or the diacylglycerol analogue dioctanoylglycerol (DiC8) also depolarized the cells. A prior activation of protein kinase C with TPA or DiC8 abolished the depolarizing effect of phenylephrine suggesting that they act through the same mediators. If the cells were made ideally permeable to K+ with the ionophore valinomycin, or the K+ channels had been blocked with Ba2+, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neither TPA nor phenylephrine had any effect on the 86Rb+ equilibrium potential across the cell membrane. The results suggest that the depolarizing effect of these substances could be through a blocking of K+ channels.

Animals↗
PubMed1989

Determination of GABA receptor-linked Cl- fluxes in rat cerebellar granule cells using a fluorescent probe SPQ.

Gamma-Aminobutyric acid (GABA)-induced Cl- fluxes in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl-ions. GABA and pentobarbitone increased the fluorescence of the probe when the Cl- gradient was directed outward by bathing cells, grown in the presence of GABA, in a low Cl- medium. Picrotoxin and bicuculline inhibited the response to GABA. The results suggest that SPQ is a suitable probe for measuring GABA-induced Cl- fluxes in living cells.

Animals↗
PubMed1989

Changes of the membrane potential in striatal synaptoneurosome, synaptosome and membrane sac preparations induced by glutamate, kainate and aspartate as measured with a cyanine dye DiS-C2-(5).

The effects of glutamate, kainate and aspartate on the membrane potential of striatal synaptoneurosome, synaptosome and membrane sac preparations were studied by using a potential sensitive cyanine dye DiS-C2-(5). Excitatory amino acids glutamate and aspartate had a depolarizing effect on synaptoneurosomes. 7.9 microM glutamate and 2.8 microM aspartate produced a half-maximal response. Depolarizations induced by glutamate and aspartate were dependent on the concentration of extracellular sodium ions, a maximal response occurred at around 40 mM of external Na+. Kainate induced a dual effect on synaptoneurosomes. In a standard Na+-based medium a hyperpolarization, likely due to inhibition of a presynaptic sodium-dependent glutamate uptake, predominated over a postsynaptic kainate receptor-mediated depolarization that was observed when electrogenic glutamate uptake was inhibited. This interpretation was supported by results obtained with synaptosome and membrane sac preparations. In a standard Na+-based medium kainate had a hyperpolarizing effect on synaptosomes while in the membrane sac preparation kainate induced a depolarization.

Animals↗
PubMed1989

(-)-Indolactam V activates protein kinase C and induces changes in muscarinic receptor functions in SH-SY5Y human neuroblastoma cells.

The effects of a synthetic protein kinase C (PKC) activator, (-)-indolactam V (ILV), were studied in SH-SY5Y human neuroblastoma cells. (-)-ILV induced a translocation of PKC from cytosol to plasma membrane and displaced 3H-phorbol dibutyrate binding in the micromolar range. In addition, (-)-ILV caused a decreased sensitivity of cells to muscarinic agonist-induced Ca2+ mobilization measured with quin-2 and induced a down-regulation of cell surface muscarinic receptors. All the changes induced by (-)-ILV were similar in magnitude to those seen with the phorbol ester tetradecanoyl phorbol acetate (TPA). The results suggest that (-)-ILV is a full activator of PKC and a promising alternative to phorbol esters in studies on mechanism of actions of PKC.

Calcium↗
PubMed1989

Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells.

Changes in membrane potential and cytosolic free Ca2+ concentrations, [Ca2+]i, in response to L-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K+ concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4-(3)). The [Ca2+]i as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K+ and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K+ induced rise in [Ca2+]i but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca2+ in response to glutamate receptor agonists is primarily due to Ca2+ influx through receptor-coupled ion channels.

Animals↗