Elsevier

Brain Research

Volume 737, Issues 1–2, 21 October 1996, Pages 175-187
Brain Research

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

https://doi.org/10.1016/0006-8993(96)00728-7Get rights and content

Abstract

To assess the role of κ-opioid receptors in astrocyte development, the effect of κ-agonists on the growth of astroglia derived from 1–2-day-old mouse cerebra was examined in vitro. κ-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of κ-receptor activation on intracellular calcium ([Ca2+]i) homeostasis and DNA synthesis. On days 6–7, as many as 50% of the astrocytes displayed κ-receptor (KA8) immunoreactivity or exhibited increases in [Ca2+]i in response to κ-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 κ-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were κ-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 κ-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 κ-opioid receptors in vitro, and suggest that the activation of κ-receptors mobilizes [Ca2+]i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing κ-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.

Introduction

Endogenous opioid peptides and receptors are present during development and can modify nervous system maturation 23, 27, 72. During development, endogenous opioid neuropeptides typically act by inhibiting the genesis of neurons and glia 23, 27, 65, 79. Pathophysiological changes in the endogenous opioid system may contribute to sudden infant death syndrome, autism or self-destructive behavior [19]. Furthermore, opiate drugs with abuse liability, such as heroin or morphine, also inhibit neural development presumably by disrupting the endogenous opioid system 23, 27. Children born to opioid addicted mothers display neurobehavioral deficits which are directly or indirectly caused by prenatal or perinatal exposure to opiates 12, 30.

The endogenous opioid system consists of multiple types of opioid receptors (e.g., μ, δ and κ) as well as a variety of endogenous opioid peptides (i.e., proopiomelanocortin, proenkephalin, and prodynorphin). μ-, δ-, and κ-opioid receptors serve many distinct functions in the adult. These receptors have been characterized pharmacologically and more recently at the molecular level 16, 44, 53, 62. Despite the importance of the endogenous opioid system in development, no single opioid receptor type has been exclusively linked to growth [27]. Moreover, in addition to the traditional μ, δ and κ-opioid receptor types 34, 35, there are putative opioid receptor types which are present during development and which may affect growth 5, 78. It is uncertain whether these immature receptors may represent novel opioid receptor types or subtypes. Thus, although multiple opioid receptor types exist, the role of the individual types in growth has not been adequately assessed.

Recent findings suggest that astrocytes are important targets of opioid action. Astrocytes express opioid binding sites [38]and opioids affect astroglial function 38, 76, including glycogen metabolism [48], cyclic AMP formation [14]and Ca2+ homeostasis 15, 28, 67. During development, not only do astrocytes themselves express endogenous opioid peptides 25, 43, 60, 61, 63, but opioids and/or opiate drugs inhibit the proliferation of flat, polyhedral (type 1) astrocytes 26, 64, 65, 66.

Despite numerous effects of opioids on astroglial function, little is known about the particular opioid receptor type(s) or the cellular mechanisms by which opioids act. Previous studies suggested that κ-opioid receptors are expressed by many developing astrocytes in vitro 14, 15, 67, and that κ receptors can affect astrocyte differentiation through a Ca2+-dependent mechanism [67]. The onset of κ-opioid receptor expression occurs early during development (by mid-gestation in rodents) 34, 35, 54. Moreover, dynorphins — a class of endogenous opioid peptides which have high affinity for κ-opioid receptors — also are expressed in the brain prenatally 34, 35. In addition to dynorphins, several of the intermediate/partially processed peptide products of proenkephalin are also expressed during early development. Recent findings suggest that proenkephalin-derived peptides can also selectively activate κ-opioid receptors, in addition to their action at δ sites [33](review 34, 35). This collective evidence prompted us to further assess the potential role of this receptor type in astrocyte development. Our results indicate that κ-opioid receptors are expressed by a subpopulation of astrocytes during development in vitro and that the activation of κ receptors inhibits cell division.

Section snippets

Materials

nor-Binaltorphimine was obtained from RBI (Research Biochemicals, Inc., Natick, MA)[68]. (−)-Naloxone was a gift from Dr. Joel G. Whitney, E.I. DuPont (Wilmington, DE). trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate (U50,488H) and [(5α,7α,8β)-(−)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]bezeneacetamide (U69,593)[32]were obtained through Dr. Paul Hillary (NIDA). Nifedipine and thapsigargin were both obtained from Sigma (St. Louis,

Results

After 3 days in vitro, 95–97% of the flat, polyhedral cells in our mixed-glial cultures were GFAP immunoreactive. These flat, polyhedral cells do not express A2B5 antigenicity and are morphologically similar to ‘type 1’ astrocytes 22, 26(Stiene-Martin, unpublished) described by other investigators [50], and are referred to as type 1 astrocytes in the remainder of the present study. We have previously found that opioids affect the development of type 1 astrocytes, but not A2B5 immunoreactive

Discussion

Our results show that two phenotypically distinct populations of astrocytes can be identified based on whether or not they express κ-opioid receptors. In type 1 astrocyte cultures in which a large percentage of cells expressed κ-receptors, κ-agonists increased [Ca2+]i and inhibited astrocyte proliferation. In addition, age-related differences in the proportion of κ- to non-κ-expressing cells were striking. A large proportion of astrocytes expressed κ-opioid receptors during the first week in

Acknowledgements

The authors wish to thank Carol Turbek, Rong Zhou, Chrystal C. Godleske, and S. Eric Ryan for technical assistance. Supported by DA 06204 and Equipment Grants from NIDA, and by the Univ. Kentucky Medical Center.

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    Present address: Department of Pharmacology, University of North Carolina, CB 7365, FLOB 1138, Chapel Hill, NC 27599-7365, USA.

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