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Biomedical subjects

I Holopainen

Publications and source records attributed to I Holopainen.

At least 37 records · Page 2Linked to original sources
PubMed1991

Ethanol-induced Cl- flux in rat cerebellar granule cells as measured by a fluorescent probe.

Cl- fluxes through the GABAA receptor gated ion channels 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. The cells were bathed in a low Cl- medium so that the Cl- gradient was directed outward. Ethanol increased the SPQ fluorescence indicating a decrease in intracellular Cl- due to Cl- efflux. Picrotoxin inhibited the effect at low concentrations of ethanol (less than 50 mM) in a concentration dependent manner. The effects of ethanol were potentiated at low concentrations (less than 10 microM) of gamma-aminobutyric acid (GABA), but inhibited at higher concentrations (0.3-2.0 mM). The results support the hypothesis that ethanol may act via the GABAA receptor gated ion channel. The results also suggest that SPQ is a suitable probe for measuring GABAA receptor-coupled Cl- fluxes through the GABAA receptor-gated channels in living cells.

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↗
PubMed1990

Single-channel and whole-cell currents in rat cerebellar granule cells.

The patch-clamp technique was used to study both whole-cell and single-channel currents in cultured rat cerebellar granule cells. In whole-cell recordings under voltage-clamp conditions 3 types of current were found; a transient inward sodium current and a transient and a sustained outward potassium current. Single-channel currents were recorded from both inside-out and outside-out membrane patches. Three types of potassium channel were identified; two non-inactivating channels with unit conductances of 160 and 60 pS and one inactivating channel of 20 pS. No calcium currents were detected.

Action Potentials↗
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

Depolarization of the neuronal membrane caused by cotransport of taurine and sodium.

C 1300 neuroblastoma cells were cultured and used to study the effect of sodium dependent taurine transport on the membrane potential. Measuring net accumulation of taurine and the depolarization caused by externally applied taurine, we found both processes become active at an external concentration of taurine of 1 mM or more. Net accumulation had Km of 13 mM and a Vmax of 126 nmol x mg of protein-1 x min-1. The taurine induced depolarization of the neuroblastoma cell was parallelled by a 25 per cent decrease in its membrane impedance. The transport of taurine, the depolarization caused by taurine and the effect of taurine on the membrane impedance, all, had a similar dependence on the external sodium concentration. Our results on the depolarizing cotransport between taurine and sodium at the neuronal membrane, may illustrate an additional mechanism for the control of the electrical activity of neuronal cells.

Cell Line↗
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

D-aspartate release from cerebellar astrocytes: modulation of the high K-induced release by neurotransmitter amino acids.

The properties of D-aspartate release were studied in cerebellar astrocytes (14-15 DIV) in primary cultures in the rat. The spontaneous release of D-aspartate from astrocytes was fast, being further enhanced in Na- and Ca-free (EDTA-containing) media. Kainate, quisqualate, D-aspartate and L-glutamate stimulated the release, whereas L-glutamatediethylester was inhibitory. The release was enhanced by veratridine and high K (50 mM). Substitution of chloride by acetate in the experimental medium did not change the basal release but slightly decreased the potassium-induced release, indicating that the high K-induced D-aspartate release is primarily due to depolarization of cells. The K-stimulated release was independent of extracellular Ca2+ and potentiated by kainate and quisqualate. The effect of kainate was reduced by kynurenate, and that of quisqualate by L-glutamatediethylester. Glycine, taurine and GABA were equally effective in depressing the stimulated release of D-aspartate. The inhibition of GABA could be blocked by GABA antagonists. The results suggest that inhibitory amino acids may be involved in the regulation of glutamate release from cerebellar astrocytes. A further implication is that cerebellar astrocytes possess functional glutamate receptors of kainate and quisqualate subtypes.

Amino Acids↗
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

Properties of single potassium channels in cultured primary astrocytes.

The patch-clamp technique was used to characterize the single channel ion currents in primary cultures of rat astrocytes. The most dominant channel type, which was found in over half of the inside-out membrane patches, was a potassium channel. The measured reversal potential was -67 mV, which is close to the calculated Nernst potential for potassium ions (-80 mV). These potassium channels activated with bursts of very brief openings. Once activated the channels did not inactivate. The measured probabilities of the channels to be closed showed at least 3 different modes of channel behaviour: one voltage-independent and two voltage-dependent modes. During each activity-mode a 'main' conductance level plus two other conductance levels were observed. In some recordings a pronounced outward rectification could also be seen.

Adenosine Triphosphate↗
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↗
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

Uptake and release of glycine in cerebellar granule cells and astrocytes in primary culture: potassium-stimulated release from granule cells is calcium-dependent.

The properties of [3H]glycine uptake and release were studied with cerebellar granule cells, 7-9 days in vitro, (DIV) and astrocytes, 14-15 DIV, in primary cultures. The uptake of glycine in both cell types consisted of a saturable high-affinity transport and nonsaturable diffusion. The transport constant (Km) and maximal velocity (V) were significantly higher in granule cells than in astrocytes. Uptake was strictly Na+-dependent and also markedly diminished in low-Cl medium. The specificity of the uptake was similar in both cell types. The spontaneous release of glycine from granule cells and astrocytes was fast. Homoexchange with extracellularly added glycine in granule cells suggests that the efflux is at least partly mediated via membrane transport sites in these cells. Kainate stimulated the release more effectively in neurons than in glial cells, the effect apparently being mediated by specific kainate-sensitive receptors in both cell types. The release was enhanced by veratridine and by depolarization of cell membranes by high K (50 mM) in both neurons and astrocytes. The potassium-stimulated release was partially Ca-dependent in neurons but Ca-independent in glial cells. The results suggest a functional role for glycine in both cerebellar astrocytes and glutamatergic granule cells.

Animals↗
PubMed1989

Release of taurine from cultured cerebellar granule cells and astrocytes: co-release with glutamate.

The properties of the release of preloaded [3H]taurine and endogenous taurine were studied with cultured cerebellar granule cells (7-8 days in vitro) and astrocytes (14-15 days in vitro) from the rat. The spontaneous release of taurine from both cell types was slow. The release from both neurons and astrocytes was significantly enhanced by 0.1 mM veratridine, the stimulatory effect being more pronounced in granule cells than in astrocytes. No homo or heteroexchange with extracellularly added taurine or its structural analogues could be detected, suggesting that the efflux is probably not mediated via the membrane transport sites. Kainate stimulated the release more from granule cells than from astrocytes, the effect apparently being mediated by kainate-sensitive receptors. Depolarization of cell membranes by 50 mM K+ induced co-release of endogenous taurine and glutamate from both cell types. Preloaded [3H]taurine was readily released from astrocytes by potassium stimulation. Stimulated release occurred from granule cells if they had been cultured for 4 days with the label but not from the cells preloaded for only 15 min.

Animals↗
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↗