Brain Research 943 (2002) 191–201 www.elsevier.com / locate / bres Research report Identification of second messengers that induce expression of functional gap junctions in microglia cultured from newborn rats a a a b ˜ ´ ´ ´ ´ Agustın D. Martınez , Eliseo A. Eugenın , Marıa C. Branes , Michael V.L. Bennett , a,b, * ´ Juan C. Saez a ´ ´ ´ Departamento de Ciencias Fisiologicas, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Alameda 340, Santiago, Chile b Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Accepted 14 March 2002 Abstract The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein 21 connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53–66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Signal transduction: gene expression Keywords: Brain macrophage; Dye coupling; Calcium; Protein kinase C; Connexin43 1. Introduction their immune phenotype. Several, but not all, features of the activated state can be induced in vitro by treating Microglia are brain macrophages and are the main microglia with proinflammatory agents, such as bacterial immune effectors of the central nervous system [16,19]. lipopolyssacharide [1], colony stimulating factor-1 [38], They rapidly transform from a quiescent to an activated complement 5a [31], amyloid beta protein [29], tumor phenotype in response to a wide range of injuries [19]. necrosis factor-a (TNF-a) and interferon g (IFN-g) Various degenerative diseases of the central nervous [2,40,12]. While protein kinase C (PKC)-dependent path- system are also characterized by the presence of numerous ways mediate some of the effects elicited by TNF-a, activated microglia [16,24,36]. IFN-g [2], amyloid beta protein [29] and colony stimulat- The process of microglia activation is characterized by ing factor-1 [38], early and transient rises in intracellular 21 morphological changes, cell proliferation and expression of calcium concentration ([Ca ]) precedes most effects i observed in microglia treated with bacterial lipopolysac- charide [1]. *Corresponding author. Tel.: 156-2-686-2862; fax: 156-2-222-5515. ´ E-mail address: jsaez@genes.bio.puc.cl (J.C. Saez). Activated microglia communicate with one another and 0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993(02)02621-5