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Biomedical subjects

A H Mulder

Publications and source records attributed to A H Mulder.

At least 163 records · Page 9Linked to original sources
PubMed1985

Cyclic AMP facilitates the electrically evoked release of radiolabelled noradrenaline, dopamine and 5-hydroxytryptamine from rat brain slices.

The adenylate cyclase activator forskolin as well as 8-bromo-cyclic AMP enhanced the electrically evoked release of 3H-noradrenaline and 3H-5-hydroxytryptamine from superfused rat neocortical slices and that of 3H-dopamine from neostriatal slices with comparable EC50's of about 0.5 and 50 microM, respectively, without affecting spontaneous tritium efflux. The phosphodiesterase inhibitor ZK 62771 (3-100 microM) also enhanced 3H-noradrenaline and 3H-dopamine release but slightly reduced 3H-5-hydroxytryptamine release. However, this drug profoundly enhanced spontaneous tritium release in the latter case. The facilitatory effect of forskolin (0.3 microM) on the release of the amine neurotransmitters was potentiated in the presence of ZK 62771 (30 microM). Therefore, cyclic AMP appears to exert a general facilitatory effect on the release of these biogenic amines from central nerve terminals.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Direct morphological and functional examination of murine pluripotent hemopoietic stem cells.

Pluripotent hemopoietic stem cells (PHSC) were isolated from adult mouse bone marrow by a combination of equilibrium density centrifugation and light-activated cell sorting for WGA-positive and H-2k antigen-positive cells. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 days and 6.6 colonies per 100 cells at 12 days after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony forming unit-spleen) equalled 135 (range, 90-230; n = 15). Enrichment for the cell type that provides radioprotection was equal to 180 +/- 70, indicating that PHSC and CFU-S are identical. Evidence is provided that the spleen seeding efficiency (the f-factor) of these cells was 0.10 and, therefore, that the average purity of the sorted PHSC was 65% (range in 15 experiments, 35-110%). The sorted cells were all in the G0 or G1 phase of the cycle. They appeared to be undifferentiated blasts by morphological criteria, the majority of the cells being similar in structure to the PHSC previously identified in less purified concentrates. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. Thirty percent of the sorted cells could be induced to form progeny in vitro. This procedure facilitates direct examination of the first events of hemopoietic regulation.

Animals↗
PubMed1985

Evidence for a presynaptic adenylate cyclase system facilitating [3H]norepinephrine release from rat brain neocortex slices and synaptosomes.

The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced [3H]norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of [3H]norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When [3H]norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the Ca2+ channel blocker Cd2+ and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of [3H]norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals.

Adenylyl Cyclases↗
PubMed1985

Effect of surface antigen labeling on spleen colony formation: comparison of the indirect immunofluorescence and the biotin-avidin methods.

Indirect immunofluorescence techniques for labeling cell surface antigens on murine pluripotent hemopoietic stem cells often result in a reduction of CFU-S numbers. This phenomenon was investigated by comparing indirect immunofluorescence and biotin-avidin methods using anti-T200 and anti-H-2Kk monoclonal antibodies. Mouse bone marrow cells treated with these monoclonal antibodies, alone or in combination with fluorescein conjugates of rabbit antirat or goat antimouse immunoglobulins, respectively, showed reduced numbers of CFU-S. The reduction in CFU-S numbers by anti-H-2Kk antibodies was dependent on the concentration of antibody and on the antigen density on the cells. Near complete CFU-S recovery was obtained with biotin-labeled antibodies and avidin-fluorescein isothiocyanate. The CFU-S recovery obtained was higher with higher numbers of biotin moieties per antibody molecule. Biotinylation itself did not influence the antibody binding properties. The protective effect was independent of the avidin-FITC concentration. Injection of carrageenan, an agent known to block macrophage activity, prevents the reduction of CFU-S recovery caused by anti-H-2Kk antibody treatment. The biotin-avidin procedure permits the measurement of antigen density on pluripotent stem cells through flow cytometry and sorting and full recovery of CFU-S in the in vivo assay.

Animals↗
PubMed1985

Thymus regeneration by bone marrow cell suspensions differing in the potential to form early and late spleen colonies.

We have determined the thymus-repopulating capacity of purified hemopoietic stem cells, bone marrow cells from mice injected four days previously with 5-fluorouracil (5-FUBM), and bone marrow cells cultured in the presence of stem-cell-activating factor (SAF; SAFBM). SAF is identical to interleukin 3 (IL-3). Purified stem cells are more enriched in day-12 CFU-S than in day-8 CFU-S. 5-FUBM consists of CFU-S that give rise to late (day-12) spleen colonies. SAFBM contains predominantly CFU-S that give rise to early spleen colonies (days 6-8). There is also a net increase in the number of spleen colony-forming units (CFU-S) in these cultures. Thymus regeneration after transplantation with either purified stem cells or 5-FUBM was delayed approximately three days as compared with that after transplantation with normal bone marrow cells. This delay can be ascribed to the absence of prothymocytes in these preparations. Thymus regeneration by SAFBM was delayed approximately ten days as compared with that after transplantation with normal bone marrow cells. The most likely explanation of these results is as follows. The prothymocytes in normal bone marrow produce a relatively limited offspring in the thymus soon after transplantation. This is rapidly replaced by the offspring of newly formed prothymocytes, the results of differentiation of the pluripotent stem cells. These stem cells also give rise to late spleen colonies. Stem cells that give rise to early spleen colonies appear to have lost the capacity for differentiation into the T-cell lineage.

Animals↗
PubMed1984

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant.

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.

Animals↗
PubMed1984

Presynaptic opiate receptor- and alpha 2-adrenoceptor-mediated inhibition of noradrenaline release in the rat brain: role of hyperpolarization?

The inhibitory actions of exogenous noradrenaline (1 microM) and clonidine (1 microM) as well as well as of the opiate receptor agonists morphine (1 microM) and [D-Ala2,D-Leu5]enkephalin (DADLE, 1 microM) on the potassium-induced Ca2+-dependent release of [3H]noradrenaline from superfused rat brain cortex slices were independent of the degree of depolarization when release was effected by 15 or 56 mM K+ for 5 min. The non-depolarization-dependent release of [3H]noradrenaline induced by exposing the slices for 5 min to medium with NaCl replaced by LiCl was only partially Ca2+-dependent and was not inhibited by the Ca2+ antagonist Cd2+ (50 microM). This release was strongly inhibited by morphine, DADLE, exogenous noradrenaline and clonidine at 1 microM concentrations both in the presence and the absence of extracellular Ca2+. Together with other data in the literature these results strongly suggest that opiate receptors and alpha 2-adrenoceptors located on noradrenergic axonal varicosities, unlike those located on cell bodies, do not primarily mediate hyperpolarization of the neuronal membrane. Instead the activation of these presynaptic receptors causes a reduction of Ca2+ availability for, or the utilization of Ca2+ by the secretion process upon invasion of an action potential.

Animals↗
PubMed1984

Isolation of murine pluripotent hemopoietic stem cells.

A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

Animals↗
PubMed1984

Presynaptic alpha 2- and postsynaptic beta-adrenoceptor sensitivity in slices of rat neocortex after chronic treatment with various antidepressant drugs.

The effect of chronic (4 weeks) treatment of rats with the antidepressant drugs desipramine, maprotiline, chlorimipramine, zimelidine or iprindol on the sensitivity of presynaptic alpha 2- and postsynaptic beta-adrenoceptors in neocortical slices was investigated. Acute (1 day) treatment with the antidepressants did not affect the efflux of cyclic-AMP induced by isoprenaline (1 microM) from neocortical slices, while after chronic treatment the efflux of cyclic AMP was consistently reduced. Following acute administration the electrically-evoked release of [3H]noradrenaline (NA) from radiolabelled cortical slices remained unchanged. Upon chronic treatment with desipramine the release of [3H]NA was enhanced by about 45%. In contrast, after chronic treatment with maprotiline or chlorimipramine the electrically-evoked release of [3H]NA was not affected, whereas release was even slightly reduced after chronic administration of zimelidine or iprindol. In all cases, however, a similar inhibitory effect of exogenous NA (0.1 microM) on the release of [3H]NA was found. These data indicate that the desensitization of postsynaptic beta-adrenoceptors in rat brain after chronic treatment with antidepressant drugs is not paralleled by a reduction of presynaptic alpha 2-adrenoceptor sensitivity.

Adrenergic alpha-Antagonists↗
PubMed1983

3-PPP antagonizes the in vitro effects of D-1 and D-2 dopamine receptor agonists in rat neostriatum.

3-PPP has been reported to be a selective dopamine autoreceptor agonist, a conclusion based on in vivo studies. In rat neostriatum in vitro however, 3-PPP antagonized concentration-dependently the stimulation of cyclic AMP efflux caused by the D-1 receptor agonist SKF 38393 and the inhibition of K+-induced [3H]acetylcholine release caused by the D-2 receptor agonist LY 141865. 3-PPP alone did not affect [3H]acetylcholine release or cyclic AMP efflux. When interpreting the effects of 3-PPP in vivo dopamine receptor antagonism should be considered.

Acetylcholine↗
PubMed1983

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors.

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.

4-Aminopyridine↗
PubMed1983

3H-noradrenaline release from rat neocortical slices in the absence of extracellular Ca2+ and its presynaptic alpha 2-adrenergic modulation. A study on the possible role of cyclic AMP.

In Ca2+ -free EGTA-containing medium veratrine (3-25 microM) concentration-dependently enhanced the efflux of 3H-noradrenaline from (radiolabelled) rat neocortical slices. Clonidine (1 microM) inhibited and phentolamine (3 microM) enhanced veratrine-induced 3'-noradrenaline release and the modulatory effects were inversely related to the veratrine concentration used. Dibutyryl-cyclic AMP, 8-Bromo-cyclic AMP (10 microM--3 mM) and the adenylate cyclase activators NaF (2 mM) and forskolin (10 microM) enhanced 3H-noradrenaline release induced by 3 microM veratrine, but had no effect on spontaneous tritium efflux. In the presence of these drugs the modulatory effects of clonidine and phentolamine on 3H-noradrenaline release were reduced as expected from the enhanced efficacy of veratrine. In contrast to these drugs the selective cyclic AMP-phosphodiesterase inhibitor ZK 62771 reduced veratrine (3 microM)-induced 3H-noradrenaline release in Ca2+ -free medium. In the presence of 1.2 mM Ca2+, 3H-noradrenaline release induced by 13 mM K+ was also inhibited. However, when 3H-noradrenaline release was effected in the presence of tetrodotoxin (0.3 microM) or by electrical field-stimulation (1 Hz), ZK 62771 slightly but significantly enhanced the release. It is postulated that cyclic AMP is involved in the secretion process in central noradrenergic varicosities and that presynaptic alpha2-adrenoceptors upon activation inhibit the secretion process through an inhibition of a presynaptically located adenylate cyclase.

Animals↗
PubMed1983

[3H]noradrenaline release from brain slices induced by an increase in the intracellular sodium concentration: role of intracellular calcium stores.

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.

Animals↗
PubMed1983

The purported dopamine agonist DPI inhibits [3H]noradrenaline release from rat cortical slices but not [3H]dopamine and [14C]acetylcholine release from rat striatal slices in-vitro.

The effects of the purported dopamine (DA) receptor agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) upon the in-vitro K+-induced release of [3H]DA and [14C]acetylcholine from rat neostriatal slices, and of [3H]noradrenaline from rat neocortical slices have been investigated and compared with those of the DA receptor agonist TL-99 and the alpha-adrenoceptor agonist clonidine, respectively. The rapid decomposition of the catechol compounds DPI and TL-99 in the Krebs-Ringer bicarbonate superfusion medium was shown to be inhibited by both the chelating agent EDTA and the reducing agent ascorbic acid. The results suggest that in-vitro DPI is unable to stimulate striatal DA receptors, whereas it is effective in stimulating cortical alpha 2-adrenoceptors (EC50 = 61 nM). It is concluded that DPI should be considered as a mixed alpha 1/alpha 2-adrenoceptor agonist and that the designation of DPI as a DA receptor agonist should be abandoned.

Acetylcholine↗
PubMed1982

Stimulation of D2-dopamine receptors in rat neostriatum inhibits the release of acetylcholine and dopamine but does not affect the release of gamma-aminobutyric acid, glutamate or serotonin.

D1-dopamine receptor stimulation did not affect the K+-induced release of [3H]GABA, [3H]glutamate, [3H]serotonin, [3H]dopamine and [14C]acetylcholine from slices of rat neostriatum. D2-dopamine receptor stimulation did not change the release of [3H]GABA, [3H]glutamate and [3H]serotonin, but inhibited the release of both [3H]dopamine and [14C]acetylcholine; this inhibition was antagonized by (-)-sulpiride. The release-inhibiting dopamine autoreceptors and the post synaptic dopamine receptors mediating the inhibition of acetylcholine release appear to be pharmacologically similar and can be classified as D2-receptors.

Acetylcholine↗