Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.

Background-Prostaglandin E 2 (PGE 2 ) plays a major role both in maintaining patency of the fetal ductus arteriosus and in closure of the ductus arteriosus after birth. The rate-limiting step in PGE 2 signal termination is PGE 2 uptake by the transporter PGT. Methods and Results-To determine the role of PGT in ductus arteriosus closure, we used a gene-targeting strategy to produce mice in which PGT exon 1 was flanked by loxP sites. Successful targeting was obtained because neither mice hypomorphic at the PGT allele (PGT Neo/Neo) nor global PGT knockout mice (PGT Ϫ/Ϫ ) exhibited PGT protein expression; moreover, embryonic fibroblasts isolated from targeted mice failed to exhibit carrier-mediated PGE 2 uptake. Although born in a normal mendelian ratio, no PGT Ϫ/Ϫ mice survived past postnatal day 1, and no PGT Neo/Neo mice survived past postnatal day 2. Necropsy revealed patent ductus arteriosus with normal intimal thickening but dilated cardiac chambers. Both PGT Neo/Neo and PGT Ϫ/Ϫ mice could be rescued through the postnatal period by giving the mother indomethacin before birth. Rescued mice grew normally and had no abnormalities by gross and microscopic postmortem analyses. In accordance with the known role of PGT in metabolizing PGE 2 , rescued adult PGT Ϫ/Ϫ mice had lower plasma PGE 2 metabolite levels and higher urinary PGE 2 excretion rates than wild-type mice. Conclusion-PGT plays a critical role in closure of the ductus arteriosus after birth by ensuring a reduction in local and/or circulating PGE 2 concentrations. (Circulation. 2010;121:529-536.)

Prostaglandins mediate autacrine and paracrine signaling over short distances. We used the renal collecting duct as a model system to test the hypothesis that local control of prostaglandin signaling is achieved by expressing inactivation in the same cell as synthesis. Immunocytochemical studies demonstrated that renal collecting ducts in situ express the prostaglandin (PG) synthesis enzyme, cyclooxygenase-1 (COX-1), as well as both components of prostaglandin metabolic inactivation, i.e. the prostaglandin uptake carrier prostaglandin transporter (PGT) and the enzyme 15-hydroxyprostaglandin dehydrogenase. We characterized this system further using the collecting duct cell line Madin-Darby canine kidney (MDCK), which retains COX-2 and prostaglandin dehydrogenase expression but which has lost PGT expression. When we reintroduced PGT, it was correctly sorted to the apical membrane where it altered the sidedness of prostaglandin E2 (PGE2) release, a process we call "vectorial release via sided reuptake." Importantly, although COX-2 and prostaglandin dehydrogenase are expressed in the same MDCK cell, they must be compartmentalized because even in the presence of excess dehydrogenase newly synthesized PGE2 is released largely un-oxidized. However, when PGE2 undergoes first release and then PGT-mediated reuptake, significant oxidation takes place, suggesting that PGT imports PGE2 into the prostaglandin dehydrogenase compartment. Our data are consistent with a new model that offers significant new mechanisms for the fine control of eicosanoid signaling.Prostaglandins (PGs) 1 represent an extreme example of context-dependent autacrine or paracrine signaling. A single type of prostaglandin, PGE2, can activate any of four receptor subtypes (EP 1 , EP 2 , EP 3 , and EP 4 ) so as to mediate changes in physiological function as diverse as gastric acid secretion, body temperature, intraocular pressure, blood pressure, and airway reactivity (1-3).Even within a single organ, such as the kidney, PGE2 has diverse effects including afferent arteriolar vasodilatation, reduction of NaCl resorption by the thick ascending limb of Henle, vasodilatation of medullary vasa rectae, and inhibition of osmotic water flow in the cortical collecting duct (2). On a smaller scale, the renal collecting expresses luminal EP 4 receptors, activation of which increases Na ϩ reabsorption and increases water reabsorption, and basolateral EP 1 receptors, activation of which signals the opposite effects (4, 5). Clearly, to achieve the requisite fidelity in PGE2 signaling, rapid inactivation must occur.PG inactivation involves active uptake into the cell followed by cytoplasmic oxidation (6). Our laboratory identified the prostaglandin transporter PGT (Slc21a2; oatp2A1) (7), which is the lead candidate for the uptake step. Targeted deletion of mouse PGT results in death at post-natal day 1, most likely the result of an inability to inactivate circulating PGE2. 2 The enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH) has been extensively characterized by ot...

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