Karl E.O. Åkerman

Ionic Dependence of Membrane Potential and Glutamate Receptor‐Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS‐C2‐(5)

Abstract: Membrane potentials of particles present in a sub‐cellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absor‐bance 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 ab‐sorbance 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 pic‐rotoxin, 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 neurotransmit‐ter receptor‐linked membrane potential changes with optical probes of membrane potential.

Coupling of human α2-adrenoceptor subtypes to regulation of cAMP production in transfected S115 cells

Stable S115 mouse mammary tumour cell lines, expressing separately alpha 2A-C10, alpha 2B-C2 and alpha 2C-C4 adrenoceptors were used to compare the receptor binding properties of alpha 2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 microM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the alpha 2A-C10 subtype, smaller in alpha 2C-C4, and minimal in alpha 2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the alpha 2B-C2 subtype (35%) than in the alpha 2A-C10 and alpha 2C-C4 subtypes (50-70%). After treatment of cells expressing alpha 2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by alpha 2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) alpha 2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected S115 cells, (2) all three alpha 2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in S115 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in S115 cells is different among the subtypes so that the alpha 2A-C10 subtype is coupled with high efficacy but with low sensitivity, the alpha 2B-C2 subtype with low efficacy but high sensitivity, and the alpha 2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least alpha 2B-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through Gs.

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.

Effect of Ethanol on γ-Aminobutyric Acid and Glycine Receptor-Coupled Cl− Fluxes in Rat Brain Synaptoneurosomes

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.

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.

Staurosporine induces a neuronal phenotype in SH-SY5Y human neuroblastoma cells that resembles that induced by the phorbol ester 12-O-tetradecanoyl phorbol-13 acetate TPA)

Treatment of SH‐SY5Y human neuroblastoma cells with the protein kinase inhibitor staurosporine, induced both morphological and functional differentiation in these cells. The effects of staurosporine were comparable to those induced by the protein kinase C (PKC) activator. 12‐O tetradecanoyl phorbol 13‐acetate (TPA), with respect to induction of neuronal differentiation, i.e. neurite outgrowth, inhibition of DNA synthesis, induction and down‐regulation of c‐myc protein expression, induction of mRNA for both neuropeptide Y (NPY) and growth associated protein 43 (GAP‐43) and stimulation of tyrosine hydroxylase expression. Staurosporine failed to translocate PKC to the membrane fraction or to stimulate phosphorylation of the endogenous PKC substrate M r 80,000 (p8O). Instead, staurosporine inhibited TPA‐induced phosphorylation of p80.

α-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.

Two human α2-adrenoceptor subtypes α2A-C10 and α2B-C2 expressed in Sf9 cells couple to transduction pathway resulting in opposite effects on cAMP production

Two human alpha-2 adrenoceptor subtypes alpha-2A-C10 and alpha-2B-C2 expressed in Sf9 cells couple to transduction pathways resulting in opposite effects on cAMP production

Coupling of Astroglial α2-Adrenoreceptors to Second Messenger Pathways

Abstract: We have investigated which α2‐receptor subtypes are expressed in cultured cortical astroglia, and their coupling to second messengers. Binding assays using [3H]rauwolscine showed a very low number of α2 receptors in the astrocytic cultures. Treatment of cultures with dibutyryl cyclic AMP (dBcAMP) increased significantly the number of receptors. The RNase protection assay was used to investigate which receptor subtype the cells express. The α2B message was expressed at a low level in both treated and untreated cells, the levels of mRNA for the α2A/D subtype were up‐regulated significantly in cells treated with dBcAMP and no expression of mRNA for the α2C subtype was detected. The α2 agonist dexmedetomidine inhibited forskolin‐induced increases in cyclic AMP both in treated and untreated cultures in a pertussis toxin‐dependent manner. This effect was abolished by the α2‐receptor antagonist rauwolscine. Selective α2‐receptor agonists dexmedetomidine, clonidine, and UK14,304 all increased intracellular calcium only in dBcAMP‐treated cells. The antagonist rauwolscine abolished this effect. Ca2+ responses were also seen in the absence of extracellular Ca2+ and they were inhibited by the phospholipase C inhibitor U‐73122, suggesting that astroglial α2 receptors are coupled to the inositol phospholipid pathway. We therefore also tested the effect of dexmedetomidine directly on inositol 1,4,5‐trisphosphate accumulation. A significant increase was seen that was blocked by the antagonist rauwolscine and, as expected, by U‐73122. In short, the results demonstrate that the α2 receptors in astroglia are coupled to multiple second messenger pathways. They are up‐regulated in cells treated with dBcAMP, which simultaneously assume a process‐bearing morphology. If this morphological change reflects some in vivo process such as reactive gliosis, the up‐regulation of α2‐receptor expression could mean an adaptive change in astrocytic responses to a common neurotransmitter, noradrenaline.

Differential expression of two α2-adrenergic receptor subtype mRNAs in human tissues

Genetic subtypes of alpha 2-adrenergic receptors (AR) may mediate distinct physiological functions, and undergo differential cell type-specific regulation. Thus, these distinct receptor subtypes are possible targets for the development of subtype-selective drugs. We have analyzed the tissue distribution of two human alpha 2-adrenoceptor subtype gene mRNAs, alpha 2-C4 and alpha 2-C10, in normal human fetal and adult tissues. Both receptor subtype mRNAs were abundantly expressed in fetal brain and choroid plexus. In non-neural fetal tissues, alpha 2-C10 mRNA was detected in spleen, kidney, adrenal gland, and skin, while alpha 2-C4 transcripts were observed only in kidney and skin. Most regions of the adult brain also expressed both subtypes, but with marked quantitative differences. For example, cerebral cortex contained predominantly alpha 2-C10 mRNA, whereas the caudate nucleus expressed mostly alpha 2-C4 mRNA. In adult peripheral tissues, alpha 2-C10 mRNA expression was most abundant in spleen and renal cortex, and expression of alpha 2-C4 mRNA was strongest in renal cortex and medulla. These different expression patterns provide evidence for the differential regulation of the two alpha 2-adrenergic receptor genes and warrant further investigation with techniques capable of improved anatomical resolution. Regional differences in receptor subtype expression may be valuable for the development of new, subtype-selective pharmacological agents with more targeted actions compared to currently used alpha 2-adrenoceptor agonists and antagonists.