Katalin Maderspach

Endogenous opioid system in developing normal and jimpy oligodendrocytes : μ and κ opioid receptors mediate differential mitogenic and growth responses

The early development of both neurons and neuroglia may be modulated by signaling through opioid mediated pathways. Neurons and astroglia not only express specific types of opiate receptors, but also respond functionally to opioids with altered rates of proliferation and growth. The present study was undertaken to determine if opioids also modulate development of the other major CNS macroglial cell, the oligodendrocyte (OL). Using well‐characterized polyclonal antibodies specific for δ‐, κ‐, and μ‐opiate receptors, OLs grown in vitro were shown to express μ‐receptors at a very immature stage prior to expression of κ‐receptors. This developmentally regulated sequence differs from the pattern of expression in neurons and astroglia. δ‐receptors are apparently absent from cultured OLs. OLs also have physiologic responses to selective μ‐ and κ‐receptor agonists and antagonists. Exposure of relatively immature O4+ OLs to the μ‐receptor agonist PL017 [H‐Tyr‐Pro‐Phe(N‐Me)‐D‐Pro‐NH2] resulted in a significant enhancement in the rate of DNA synthesis. This effect, which was not observed in more mature MBP+ OLs, was entirely blocked by the antagonist naloxone. Although the κ‐receptor pathway appeared to be uninvolved in controlling proliferation, the κ‐receptor antagonist nor‐binaltorphimine significantly increased the size of myelin‐like membranes produced by the cultured OLs. Interestingly, OLs derived from the jimpy mouse, a mutant characterized by an almost complete lack of CNS myelin and premature death of OLs, were found to be deficient in κ‐opiate receptors. Our findings clearly show that OLs not only express specific opiate receptors, but also respond to changes in their level of stimulation in ways that could profoundly impact nervous system morphology and function. If opiate receptors are expressed by OLs in vivo, their pharmacological manipulation might provide a novel pathway for modulating OL and myelin production both during development and in demyelinated conditions. GLIA 22:189–201, 1998.© 1998 Wiley‐Liss, Inc.

Glial and neuronal opioid receptors: apparent positive cooperativity observed in intact cultured cells.

Opioid receptors were characterized in glial and neuronal homogeneous cultures of embryonic chick forebrain, using [3H]naloxone as a labelled ligand. Binding experiments were performed on intact cells. The specific binding of [3H]naloxone reached equilibrium after 1 min. The apparent dissociation constants were estimated as 0.51 nM for glial and 0.63 nM for neuronal cells. Equilibrium measurements indicated the apparent positive cooperativity of the binding, resulting in Hill coefficients of 2.61 for glial and 2.04 for neuronal cells. Competition of unlabelled naloxone for specific binding sites resulted in maximum-shape curves in glial cells if measured at low receptor occupancy. This supports the positive cooperativity of ligand binding. Opioid agonists, ethylketocyclazocine (EKC), morphine and [D-Ala2,L-Leu5]enkephalin (DALA), provoked biphasic competition curves in both cell types with a characteristic maximum at low competitor concentrations. The possible physiological role of glial opioid receptors in neuron-glia communication and the significance of cooperativity is discussed.

κ-Opioid receptor expression defines a phenotypically distinct subpopulation of astroglia: relationship to Ca2+ mobilization, development, and the antiproliferative effect of opioids

To assess the role of kappa-opioid receptors in astrocyte development, the effect of kappa-agonists on the growth of astroglia derived from 1-2-day-old mouse cerebra was examined in vitro. kappa-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of kappa-receptor activation on intracellular calcium ([Ca2+]i) homeostasis and DNA synthesis. On days 6-7, as many as 50% of the astrocytes displayed kappa-receptor (KA8) immunoreactivity or exhibited increases in [Ca2+]i in response to kappa-agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the kappa-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were kappa-opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca2+]i. Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that kappa-receptor activation increases astroglial [Ca2+]i both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca2+ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express kappa-opioid receptors in vitro, and suggest that the activation of kappa-receptors mobilizes [Ca2+]i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing kappa-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.

Muscarinic acetylcholine receptors on cultured glia cells

Muscarinic acetylcholine receptors (76 fmol/mg protein) were detected on cultured glia cells (astroblasts) from embryonic chicken brain by specific [3H]quinuclidinylbenzilate (QNB) binding at physiological conditions. The QNB binding (Kd = 9.5 x 10(-11)) to the intact cells seems to be cooperative (nH = 1.98) as shown by graphical methods.

Cellular distribution of the mRNA for the κ-opioid receptor in the human neocortex: a non-isotopic in situ hybridization study ☆

Opioid receptors (OR) provide primary interaction sites of the human brain with opiates. Presently kappa-OR mRNA expression was studied in different cortical areas (A4, A10, A17) by in situ hybridization using digoxigenin-labeled oligonucleotides and an alkaline phosphatase-mediated color reaction. kappa-OR mRNA was expressed mainly in layers II/III and V pyramidal and layer VI multiform neurons. A4 giant pyramidal and A17 giant stellate neurons stood out labeled. These findings fit in with our data on kappa-OR protein distribution. Combined cellular assessment of protein and mRNA will enable the study kappa-OR expression under physiological and pathological conditions.

β-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.

Development of β-adrenergic receptors and their function in glia-neuron communication in cultured chick brain

The beta-receptors of intact neuronal and glial cells of chick embryonic brain were studied via the specific binding of the beta-antagonist [3H]dihydro-L-alprenolol ( [3H]DHA). Cells were cultivated in either highly homogeneous or mixed populations; the neuronal cells were also grown under the influence of glial conditioned medium (GCM) or 10(-11)-10(-10) M L-norepinephrine or L-isoproterenol. The beta-receptors of both neuronal and glial cells proved to be positively cooperative (n = 2.5) and of high affinity, with a Kdapp of 98 and 44 pM, respectively. The Kdapp value was influenced only slightly by the different culture conditions. The receptor concentration was relatively low in the homogeneous neuronal and glial cultures (Bmax = 6.4 and 3.3 fmol/10(6) cells, respectively). It increased by a factor of 2-3 if development of the neuron-glia contacts in the culture was possible (mixed cultures). GCM and beta-agonists elevated the number of beta-receptors of the neuronal cells approximately 4-fold, even in the absence of glial cells. This receptor-number change was preceded by a well observable morphological differentiation. Both the morphological and the beta-receptor effects of L-norepinephrine were antagonized by L-propranolol. The beta-receptor number increased about 2-fold during a 10-day in vitro development, even in neuron-glia mixed cultures.

A Monoclonal Antibody Recognizing K‐ but Not γ‐ and δ‐Opioid Receptors

Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k‐opioid receptor binding site was generated. BALB/ c female mice were immunized with a partially purified k‐opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibodyproducing hybridomas were screened for competition with opioid ligands in a modified enzyme‐linked immunosorbent assay. The cell line KA8 secretes an IgG1 (k‐light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A‐Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity‐purified frog brain k‐opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration‐dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when γ‐and δ‐opioid binding was suppressed by 100 nM [D‐Ala2,NMe‐Phe4,Gly‐ol]‐enkephalin (DAGO) and 100 nM [D‐Ala2,L‐Leu5]‐enkephalin (DADLE), respectively, and to 100% when γ‐, δ‐, and k2‐sites were blocked by 5 γM DADLE. However, the γ‐specific [3H]DAGO and the δ‐preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the k‐ but not the γ‐ and δ‐subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes. These findings suggest homologies in the k‐opioid binding site of frog brain and rat brain as well as chick neurons, but the k‐opioid receptor subtype in the guinea pig cerebellum may be different.

Immunohistochemical localization of κ opioid receptors in the human frontal cortex

Abstract The cellular and subcellular κ opioid receptor distribution in human frontal cortex was studied using the monoclonal antibody (KA8). κ opioid receptor-like immunoreactivity was mainly localized in pyramidal neurons of layers II/III and V. In addition, some round and ovoid neurons were found immunolabeled mainly in layer VI. At the ultrastructural level the immunoprecipitate was attached to the cell membrane but was not associated with synaptic specializations. Furthermore, labeling was present in the neuronal perikarya associated with free ribosomes and in the dendrites partly decorating microtubular structures. Previous autoradiographic findings remained restricted to the level of laminar distribution. By contrast, the monoclonal antibody KA8 provides a unique tool to study the cerebral κ opioid receptor distribution on the cellular and subcellular level.

Kappa opioid receptors are expressed by interneurons in the CA1 area of the rat hippocampus: a correlated light and electron microscopic immunocytochemical study

A local GABA-system is known to have a mediatory function between several afferents and the principal cells of the hippocampus. This study examines the distribution and fine structure of kappa opioid receptor-immunoreactive elements in the CA1 subfield and reveals some new aspects concerning the structural basis of opioid-GABA interaction in the rat hippocampal formation. Kappa receptors were visualized immunocytochemically with a previously produced and characterized monoclonal antibody, the mAb KA8 (Maderspach, K., Németh, K., Simon, J., Benyhe, S., Szûcs, M., Wollemann, M., 1991. A monoclonal antibody recognizing kappa-, but not mu- and delta-opioid receptors. J. Neurochem. 56, 1897-1904). The antibody selectively recognizes the kappa opioid receptor with preference to the kappa(2) subtype. Neuronal cell bodies, proximal dendrites and occasionally glial processes surrounding neuronal perikarya were labelled in the CA1 area. The immunopositive cells were present mainly in the stratum oriens, followed by the stratum pyramidale in a rostrocaudally increasing number. Their shape was fusiform, or multipolar. Occasionally kappa receptor-immunoreactive boutons surrounding weakly immunopositive somata were also observed. Electron microscopy of immunopositive neurons showed that the DAB labelling was intensive in the perinuclear cytoplasm. The widths and electron densities of the postsynaptic densities of some axosomatic synapses were remarkably increased. Similar increase of postsynaptic densities were observable at some axodendritic and axospinous synapses. On the basis of their location and fine structural properties the labelled cells are suggested to be GABAergic inhibitory interneurons, probably belonging to the somatostatinergic sub-population. The axons of these inhibitory interneurons are known to arborize in the stratum lacunosum-moleculare where the entorhinal afferents terminate. A modulatory effect of opioids on the entorhinal input, mediated by somatostatinergic interneurons is suggested