Cell-cell communication plays a central role in the guidance of migrating neurons during the deve... more Cell-cell communication plays a central role in the guidance of migrating neurons during the development of the cerebral cortex. Neuregulins (NRGs) are essential mediators for migration and maintenance of the radial glial scaffold. We show here that soluble NRG reduces neuronal motility, causes transition of bipolar cells to multipolar ones and induces neuronal mitosis. Blocking the NRG receptor, ErbB4 results in reduction of neuron-neuron and neuron-radial glial contacts and causes an increase in neuronal motility. Blocking the radial glial metabotropic glutamate receptor 5 (mGluR5), the nonselective cation channel TRPC3, or matrix metalloproteases (MMP) results in similar effects as ErbB4 blockade. Soluble NRG counteract the changes in motility pattern. Stimulation of other radial glial G-protein coupled receptors such as muscarinic acetylcholine receptors or endothelin receptors counteract all the effect of mGluR5 blockade but not that of ErbB4, TRPC3 and MMP blockade. The results indicate that neurotransmitters and endothelins acting on radial glial G-protein coupled receptors are, via proteolytic NRG/ErbB4 activation, able to modify the migratory behavior of neurons.
Suboptimal concentrations of the anti-IL-4 receptor mAb M1 or M2 gave, in combination with IL-4, ... more Suboptimal concentrations of the anti-IL-4 receptor mAb M1 or M2 gave, in combination with IL-4, an enhanced proliferative response to the IL-4-responsive cell line D10.G4.1, compared to IL-4 alone. Increasing amounts of the M1 antibody inhibited the IL-4-dependent proliferation in a normal fashion. The enhanced IL-4-induced proliferation was only inhibited with the anti-IL-4 mAb 11B11, whereas the anti-IL-2 receptor antibodies had no effect. Addition of M1 or M2 antibodies to the IL-2-dependent cell line CTLL, known to express small amounts of IL-4 receptors and thereby also slightly responsive to IL-4, gave no enhanced IL-4-induced proliferation. Instead these antibodies were found to inhibit both the IL-4 as well as the IL-2-mediated CTLL proliferation.
The effects of glutamate, kainate and aspartate on the membrane potential of striatal synaptoneur... more 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.
International Journal of Developmental Neuroscience, Nov 16, 2006
oxidantand inflammatory-mediated diseases, including brain aging. In the present study we report ... more oxidantand inflammatory-mediated diseases, including brain aging. In the present study we report that treatment for 4 months of senescent rats with Acetylcarnitine (LAC) is able to induce heme oxygenase-1, Hsp70 and SOD-2, and that this effect is associated with up-regulation of GSH levels. In addition, by using redox proteomics, we provide a valuable insight into the mechanism of age-related protein oxidation and the effect of LAC in reducing oxidative stress associated with functional impairment in aged brains. Redox proteomics analysis of hippocampus (HP) and cerebral cortex (CX), brain regions in which all indices of oxidative modification are elevated in brain aging showed that the specific carbonyl levels of three proteins, hemoglobin, cofilin 1 and beta-actin, are significantly increased in HP of senescent rats. All specific carbonyl levels of these proteins are reduced by LAC administration in old rats. In the CX of senescent rats, the specific carbonyl levels of eight proteins: heat shock protein 70, glyoxylase 1, beta-actin, 3-mercaptopyruvate sulfurtransferase, peroxiredoxin 1, phosphoribosyl pyrophosphate synthetase 1, and fumarase, were increased. LAC administration was able to reduce the specific carbonyl levels of all these proteins which are involved in three impaired processes in aged brains: antioxidant defence, mitochondrial function and synaptic plasticity. Treatment by LAC may improve the decline of these functions and therefore LAC could be considered as a potential therapeutic agent for treatment of cognitive decline in aging. Therapeutic strategies focussing on acetylcarnitine treatment may represent a crucial mechanism of defence against free radical-induced damage of critical proteins occurring in aging brain.
Advances in Experimental Medicine and Biology, 1999
Oxidative metabolism plays a key role in glucose-induced insulin release (Ashcroft et al., 1973).... more Oxidative metabolism plays a key role in glucose-induced insulin release (Ashcroft et al., 1973). With the development of a sensitive assay for insulin it was possible to characterize the dynamics of glucose-induced insulin release from the individual isolated islet of Langerhans (Bergsten and Hellman, 1993). To evaluate the role of the oxidative metabolism for the kinetics of insulin release a method for determining oxidative metabolism with a sensitivity comparable to that available for insulin is crucial. Glucose oxidation and oxygen consumption have been used to monitor glucose-induced metabolic changes (Ashcroft et al., 1973; Hutton and Malaisse, 1980; Longo et al., 1991). These studies indicate that insulin release and cellular metabolism, oxidation in particular, are tightly linked. However, the previously used techniques lack the sensitivity required to monitor the dynamics of metabolism from individual islets. Thus analysis of the coupling or synchronization between metabolic changes and secretion is hampered. To overcome this problem we have developed a new sensitive technique to monitor oxygen tension, which allows time-resolved measurements from individual islets, and characterized the responses in pO2 following exposure to glycolytic and mitochondrial substrates.
The extracellular Ca2+ concentration has a strong influence on the function of retinal photorecep... more The extracellular Ca2+ concentration has a strong influence on the function of retinal photoreceptors. In the present study both single- and double-barrelled Ca2+-sensitive microelectrodes were used to measure the free calcium ion concentration in the extracellular fluid bathing the photoreceptors in opened excised eyes of frogs and carps. The electrode tip was localized electrophysiologically by voltage drops related to current pulses passed radially through the preparation, using the high-resistance pigment epithelium as a landmark. The measurements showed that the extracellular Ca2+ concentration is very close or identical to that prevailing in the vitreous humour, 1.0-1.1 mM in the frog retina and 1.2-1.4 mM in the carp.
The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using t... more The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.
Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with ... more Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with receptor binding, Ca++ mobilization and Northern blotting. Displacement of [3H]NMS with pirenzepine in IMR-32 cells revealed apparent binding sites with Kd values of 5 (41%) and 237 nM (59%). With 4-diphenylacetoxy-N-metylpiperidine metiodid, a similar proportion of apparent high- and low-affinity binding was obtained: 36 (Kd = 0.26 nM) and 64% (Kd = 6.3 nM), respectively. In SH-SY5Y cells, two different affinities with apparent Kd of 40 (24%) and 460 nM (76%) could be distinguished with pirenzepine, even though the Kd of the apparent high-affinity site varied markedly (variation = 8.7-96.8 nM). Inhibition of carbachol-induced Ca++ mobilization displayed high sensitivity to 4-diphenylacetoxy-N-methylpiperidine metiodid in both cell lines. IMR-32 cells displayed high sensitivity to pirenzepine, whereas the sensitivity varied between different batches of SH-SY5Y cells. DNA fragments (approximately 1000 base pairs) from SH-SY5Y DNA amplified with polymerase chain reaction were used as probes for muscarinic receptor mRNA. Northern blotting with the Hm1-specific probe gave a stronger signal for SH-SY5Y than for IMR-32, whereas the result obtained with the Hm2-probe was the opposite. Also, the Hm3 mRNA was detected in SH-SY5Y cells. The Hm4 and Hm5 transcripts were not detected in either of these cell lines.
The agonist profiles for Ca++ elevations mediated by the human alpha-2 adrenoceptor subtypes alph... more The agonist profiles for Ca++ elevations mediated by the human alpha-2 adrenoceptor subtypes alpha-2A, alpha-2B and alpha-2C were compared in the clones of Chinese hamster ovary cells expressing comparable numbers of receptors. No difference was seen between the different clones with respect to the maximum Ca++ mobilizations or the concentrations producing half-maximal stimulation in response to noradrenaline. Ca++ elevations were sensitive to phospholipase C inhibitor U-73122 (1-[6-([17beta]-3-methoxyestra-1,3, 5[10]-trien-17-yl)aminohexyl]-1H-pyrrole-2,5-dione) and pertussis toxin-pretreatment. Although noradrenaline was equally potent and active in all the clones, marked differences in the response to the other agonists were seen. UK14,304 (5-bromo-N-[4, 5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine) was a full agonist (when compared to noradrenaline) for alpha-2A and alpha-2C, D-medetomidine ([+]-[S]-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl) was a full agonist for alpha-2B and alpha-2C and oxymetazoline (3-[(4, 5-dihydro-1H-imidazol-2-yl-)methyl]-6-[1,1-dimethylethyl]-2, 4-dimethylphenol HCl) was a full agonist only for alpha-2B receptors. Clonidine (2-[2,6-dichloroaniline]-2-imidazoline HCl) was a partial agonist in all the cases; almost no response to this ligand was obtained in the alpha-2B-expressing cells. When the Ca++ responses are compared to the previously published results on cAMP inhibition in Chinese hamster ovary cells, clonidine seems to be significantly less efficacious in elevating Ca++ than in decreasing cAMP.
The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) ... more The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdCl2. This inhibition is partially relieved by an increase in the external Ca2+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 45Ca2+ influx is unaltered by CdCl2, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdCl2 (100 microM). The steady-state efflux of 45Ca2+ and the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of erythrocyte ghosts are inhibited by CdCl2 (10 microM). Thus, the mechanism behind the Cd2+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca2+ influx. We suggest that Cd2+ competes with Ca2+ for intracellular Ca2+-binding molecules, such as calmodulin, essential for the induction of cell proliferation.
Strychnine-sensitive, inhibitory glycine receptors have not until lately been considered to play ... more Strychnine-sensitive, inhibitory glycine receptors have not until lately been considered to play a significant role in neurotransmission in mammalian forebrain regions. In order to investigate the role of glycine as a neurotransmitter in brain we have measured glycine induced chloride fluxes in different adult rat forebrain areas using synaptoneurosomes and a chloride-sensitive fluorescent indicator. The results have been compared to those obtained with GABA. The synaptoneurosomes from every brain area investigated responded to both glycine and GABA with chloride fluxes in a picrotoxin sensitive manner. The effect of glycine was inhibited by strychnine, which had no effect on the GABA-induced CI-flux. Bicuculline inhibited the effect of GABA, but had no effect on the glycine-induced C1 flux. Addition of GABA did not affect the response to glycine and vice versa. The endogenous content of glycine and GABA in the synaptoneurosome preparations was about the same and synaptoneurosomes from every brain area investigated released both glycine and GABA upon depolarisation with KC1. The depolarisation induced release of both GABA and glycine was partly Ca:+-dependent and partly Ca2+-independent. These results indicate that glycine can induce inhibitory CI-fluxes distinct from GABA induced fluxes in every investigated brain area and that glycine can be released upon depolarisation.
The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (... more The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca2+ mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(+/-)-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e. , it caused Ca2+ mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca2+ levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse alpha2-adrenergic ligands also were tested. Ligands that could promote increases in Ca2+ levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the alpha2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the alpha2A-adrenoceptors in HEL 92.1.7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca2+ and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a "protean agonist" because it is capable of activating uncoupled alpha2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled alpha2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor "tone" could be adjusted, which should provide a new therapeutic principle in receptor dysfunction.
is an endogenous danger signal that is known to activate inflammatory responses in innate immune ... more is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1b. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1b and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesiclemediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.
Both phenylephrine and carbachol caused a sustained increase in Ca z+ influx and intracellular fr... more Both phenylephrine and carbachol caused a sustained increase in Ca z+ influx and intracellular free Ca "~ 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 Ca 2+. If extracellular Ca-,+ was removed by EGTA only a transient rise in cytosolic Ca 2+ 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 (DiCs) 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 Ba 2+, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neiher 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.
Cerebellar granule cell; Excitatory amino acid; Fura-2; Bisoxonol: Membrane potential: Intracellu... more Cerebellar granule cell; Excitatory amino acid; Fura-2; Bisoxonol: Membrane potential: Intracellular calcium Changes in membrane potential and cytosolic free Ca 2+ concentrations, [Ca t ~]~, 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 [Cae']~ 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 [Ca:+]~ but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca 2~ in response to glutamate receptor agonists is primarily due to Ca-'+ influx through receptorcoupled ion channels.
Chloride fluxes in Synaptoneurosomes in response to additions of γ‐aminobutyric acid, glycine, an... more Chloride fluxes in Synaptoneurosomes in response to additions of γ‐aminobutyric acid, glycine, and ethanol were measured using a chloride‐sensitive fluorescent probe 6‐methoxy‐N‐(3‐sulfopropyl) quinolinium (SPQ). The C1− gradient was directed outward by bathing cells in a medium low in C1− concentration. The Synaptoneurosomes responded to both γ‐aminobutyric acid and glycine by outflow of C1− ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of C1− 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 γ‐aminobutyric acid and glycine receptor‐linked chloride fluxes in the rat brain.
Cell-cell communication plays a central role in the guidance of migrating neurons during the deve... more Cell-cell communication plays a central role in the guidance of migrating neurons during the development of the cerebral cortex. Neuregulins (NRGs) are essential mediators for migration and maintenance of the radial glial scaffold. We show here that soluble NRG reduces neuronal motility, causes transition of bipolar cells to multipolar ones and induces neuronal mitosis. Blocking the NRG receptor, ErbB4 results in reduction of neuron-neuron and neuron-radial glial contacts and causes an increase in neuronal motility. Blocking the radial glial metabotropic glutamate receptor 5 (mGluR5), the nonselective cation channel TRPC3, or matrix metalloproteases (MMP) results in similar effects as ErbB4 blockade. Soluble NRG counteract the changes in motility pattern. Stimulation of other radial glial G-protein coupled receptors such as muscarinic acetylcholine receptors or endothelin receptors counteract all the effect of mGluR5 blockade but not that of ErbB4, TRPC3 and MMP blockade. The results indicate that neurotransmitters and endothelins acting on radial glial G-protein coupled receptors are, via proteolytic NRG/ErbB4 activation, able to modify the migratory behavior of neurons.
Suboptimal concentrations of the anti-IL-4 receptor mAb M1 or M2 gave, in combination with IL-4, ... more Suboptimal concentrations of the anti-IL-4 receptor mAb M1 or M2 gave, in combination with IL-4, an enhanced proliferative response to the IL-4-responsive cell line D10.G4.1, compared to IL-4 alone. Increasing amounts of the M1 antibody inhibited the IL-4-dependent proliferation in a normal fashion. The enhanced IL-4-induced proliferation was only inhibited with the anti-IL-4 mAb 11B11, whereas the anti-IL-2 receptor antibodies had no effect. Addition of M1 or M2 antibodies to the IL-2-dependent cell line CTLL, known to express small amounts of IL-4 receptors and thereby also slightly responsive to IL-4, gave no enhanced IL-4-induced proliferation. Instead these antibodies were found to inhibit both the IL-4 as well as the IL-2-mediated CTLL proliferation.
The effects of glutamate, kainate and aspartate on the membrane potential of striatal synaptoneur... more 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.
International Journal of Developmental Neuroscience, Nov 16, 2006
oxidantand inflammatory-mediated diseases, including brain aging. In the present study we report ... more oxidantand inflammatory-mediated diseases, including brain aging. In the present study we report that treatment for 4 months of senescent rats with Acetylcarnitine (LAC) is able to induce heme oxygenase-1, Hsp70 and SOD-2, and that this effect is associated with up-regulation of GSH levels. In addition, by using redox proteomics, we provide a valuable insight into the mechanism of age-related protein oxidation and the effect of LAC in reducing oxidative stress associated with functional impairment in aged brains. Redox proteomics analysis of hippocampus (HP) and cerebral cortex (CX), brain regions in which all indices of oxidative modification are elevated in brain aging showed that the specific carbonyl levels of three proteins, hemoglobin, cofilin 1 and beta-actin, are significantly increased in HP of senescent rats. All specific carbonyl levels of these proteins are reduced by LAC administration in old rats. In the CX of senescent rats, the specific carbonyl levels of eight proteins: heat shock protein 70, glyoxylase 1, beta-actin, 3-mercaptopyruvate sulfurtransferase, peroxiredoxin 1, phosphoribosyl pyrophosphate synthetase 1, and fumarase, were increased. LAC administration was able to reduce the specific carbonyl levels of all these proteins which are involved in three impaired processes in aged brains: antioxidant defence, mitochondrial function and synaptic plasticity. Treatment by LAC may improve the decline of these functions and therefore LAC could be considered as a potential therapeutic agent for treatment of cognitive decline in aging. Therapeutic strategies focussing on acetylcarnitine treatment may represent a crucial mechanism of defence against free radical-induced damage of critical proteins occurring in aging brain.
Advances in Experimental Medicine and Biology, 1999
Oxidative metabolism plays a key role in glucose-induced insulin release (Ashcroft et al., 1973).... more Oxidative metabolism plays a key role in glucose-induced insulin release (Ashcroft et al., 1973). With the development of a sensitive assay for insulin it was possible to characterize the dynamics of glucose-induced insulin release from the individual isolated islet of Langerhans (Bergsten and Hellman, 1993). To evaluate the role of the oxidative metabolism for the kinetics of insulin release a method for determining oxidative metabolism with a sensitivity comparable to that available for insulin is crucial. Glucose oxidation and oxygen consumption have been used to monitor glucose-induced metabolic changes (Ashcroft et al., 1973; Hutton and Malaisse, 1980; Longo et al., 1991). These studies indicate that insulin release and cellular metabolism, oxidation in particular, are tightly linked. However, the previously used techniques lack the sensitivity required to monitor the dynamics of metabolism from individual islets. Thus analysis of the coupling or synchronization between metabolic changes and secretion is hampered. To overcome this problem we have developed a new sensitive technique to monitor oxygen tension, which allows time-resolved measurements from individual islets, and characterized the responses in pO2 following exposure to glycolytic and mitochondrial substrates.
The extracellular Ca2+ concentration has a strong influence on the function of retinal photorecep... more The extracellular Ca2+ concentration has a strong influence on the function of retinal photoreceptors. In the present study both single- and double-barrelled Ca2+-sensitive microelectrodes were used to measure the free calcium ion concentration in the extracellular fluid bathing the photoreceptors in opened excised eyes of frogs and carps. The electrode tip was localized electrophysiologically by voltage drops related to current pulses passed radially through the preparation, using the high-resistance pigment epithelium as a landmark. The measurements showed that the extracellular Ca2+ concentration is very close or identical to that prevailing in the vitreous humour, 1.0-1.1 mM in the frog retina and 1.2-1.4 mM in the carp.
The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using t... more The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.
Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with ... more Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with receptor binding, Ca++ mobilization and Northern blotting. Displacement of [3H]NMS with pirenzepine in IMR-32 cells revealed apparent binding sites with Kd values of 5 (41%) and 237 nM (59%). With 4-diphenylacetoxy-N-metylpiperidine metiodid, a similar proportion of apparent high- and low-affinity binding was obtained: 36 (Kd = 0.26 nM) and 64% (Kd = 6.3 nM), respectively. In SH-SY5Y cells, two different affinities with apparent Kd of 40 (24%) and 460 nM (76%) could be distinguished with pirenzepine, even though the Kd of the apparent high-affinity site varied markedly (variation = 8.7-96.8 nM). Inhibition of carbachol-induced Ca++ mobilization displayed high sensitivity to 4-diphenylacetoxy-N-methylpiperidine metiodid in both cell lines. IMR-32 cells displayed high sensitivity to pirenzepine, whereas the sensitivity varied between different batches of SH-SY5Y cells. DNA fragments (approximately 1000 base pairs) from SH-SY5Y DNA amplified with polymerase chain reaction were used as probes for muscarinic receptor mRNA. Northern blotting with the Hm1-specific probe gave a stronger signal for SH-SY5Y than for IMR-32, whereas the result obtained with the Hm2-probe was the opposite. Also, the Hm3 mRNA was detected in SH-SY5Y cells. The Hm4 and Hm5 transcripts were not detected in either of these cell lines.
The agonist profiles for Ca++ elevations mediated by the human alpha-2 adrenoceptor subtypes alph... more The agonist profiles for Ca++ elevations mediated by the human alpha-2 adrenoceptor subtypes alpha-2A, alpha-2B and alpha-2C were compared in the clones of Chinese hamster ovary cells expressing comparable numbers of receptors. No difference was seen between the different clones with respect to the maximum Ca++ mobilizations or the concentrations producing half-maximal stimulation in response to noradrenaline. Ca++ elevations were sensitive to phospholipase C inhibitor U-73122 (1-[6-([17beta]-3-methoxyestra-1,3, 5[10]-trien-17-yl)aminohexyl]-1H-pyrrole-2,5-dione) and pertussis toxin-pretreatment. Although noradrenaline was equally potent and active in all the clones, marked differences in the response to the other agonists were seen. UK14,304 (5-bromo-N-[4, 5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine) was a full agonist (when compared to noradrenaline) for alpha-2A and alpha-2C, D-medetomidine ([+]-[S]-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl) was a full agonist for alpha-2B and alpha-2C and oxymetazoline (3-[(4, 5-dihydro-1H-imidazol-2-yl-)methyl]-6-[1,1-dimethylethyl]-2, 4-dimethylphenol HCl) was a full agonist only for alpha-2B receptors. Clonidine (2-[2,6-dichloroaniline]-2-imidazoline HCl) was a partial agonist in all the cases; almost no response to this ligand was obtained in the alpha-2B-expressing cells. When the Ca++ responses are compared to the previously published results on cAMP inhibition in Chinese hamster ovary cells, clonidine seems to be significantly less efficacious in elevating Ca++ than in decreasing cAMP.
The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) ... more The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdCl2. This inhibition is partially relieved by an increase in the external Ca2+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 45Ca2+ influx is unaltered by CdCl2, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdCl2 (100 microM). The steady-state efflux of 45Ca2+ and the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of erythrocyte ghosts are inhibited by CdCl2 (10 microM). Thus, the mechanism behind the Cd2+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca2+ influx. We suggest that Cd2+ competes with Ca2+ for intracellular Ca2+-binding molecules, such as calmodulin, essential for the induction of cell proliferation.
Strychnine-sensitive, inhibitory glycine receptors have not until lately been considered to play ... more Strychnine-sensitive, inhibitory glycine receptors have not until lately been considered to play a significant role in neurotransmission in mammalian forebrain regions. In order to investigate the role of glycine as a neurotransmitter in brain we have measured glycine induced chloride fluxes in different adult rat forebrain areas using synaptoneurosomes and a chloride-sensitive fluorescent indicator. The results have been compared to those obtained with GABA. The synaptoneurosomes from every brain area investigated responded to both glycine and GABA with chloride fluxes in a picrotoxin sensitive manner. The effect of glycine was inhibited by strychnine, which had no effect on the GABA-induced CI-flux. Bicuculline inhibited the effect of GABA, but had no effect on the glycine-induced C1 flux. Addition of GABA did not affect the response to glycine and vice versa. The endogenous content of glycine and GABA in the synaptoneurosome preparations was about the same and synaptoneurosomes from every brain area investigated released both glycine and GABA upon depolarisation with KC1. The depolarisation induced release of both GABA and glycine was partly Ca:+-dependent and partly Ca2+-independent. These results indicate that glycine can induce inhibitory CI-fluxes distinct from GABA induced fluxes in every investigated brain area and that glycine can be released upon depolarisation.
The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (... more The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca2+ mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(+/-)-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e. , it caused Ca2+ mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca2+ levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse alpha2-adrenergic ligands also were tested. Ligands that could promote increases in Ca2+ levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the alpha2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the alpha2A-adrenoceptors in HEL 92.1.7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca2+ and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a "protean agonist" because it is capable of activating uncoupled alpha2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled alpha2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor "tone" could be adjusted, which should provide a new therapeutic principle in receptor dysfunction.
is an endogenous danger signal that is known to activate inflammatory responses in innate immune ... more is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1b. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1b and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesiclemediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.
Both phenylephrine and carbachol caused a sustained increase in Ca z+ influx and intracellular fr... more Both phenylephrine and carbachol caused a sustained increase in Ca z+ influx and intracellular free Ca "~ 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 Ca 2+. If extracellular Ca-,+ was removed by EGTA only a transient rise in cytosolic Ca 2+ 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 (DiCs) 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 Ba 2+, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neiher 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.
Cerebellar granule cell; Excitatory amino acid; Fura-2; Bisoxonol: Membrane potential: Intracellu... more Cerebellar granule cell; Excitatory amino acid; Fura-2; Bisoxonol: Membrane potential: Intracellular calcium Changes in membrane potential and cytosolic free Ca 2+ concentrations, [Ca t ~]~, 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 [Cae']~ 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 [Ca:+]~ but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca 2~ in response to glutamate receptor agonists is primarily due to Ca-'+ influx through receptorcoupled ion channels.
Chloride fluxes in Synaptoneurosomes in response to additions of γ‐aminobutyric acid, glycine, an... more Chloride fluxes in Synaptoneurosomes in response to additions of γ‐aminobutyric acid, glycine, and ethanol were measured using a chloride‐sensitive fluorescent probe 6‐methoxy‐N‐(3‐sulfopropyl) quinolinium (SPQ). The C1− gradient was directed outward by bathing cells in a medium low in C1− concentration. The Synaptoneurosomes responded to both γ‐aminobutyric acid and glycine by outflow of C1− ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of C1− 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 γ‐aminobutyric acid and glycine receptor‐linked chloride fluxes in the rat brain.
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Papers by Karl Åkerman