### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

/pdf/receptors-for-purines-and-pyrimidines-56f78tnpew.pdf

Receptors for Purines and Pyrimidines

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

/pdf/atp-mediates-rapid-microglial-response-to-local-brain-injury-4capw6j3mj.pdf

ATP mediates rapid microglial response to local brain injury in vivo

P2X 7 receptors are ATP‐gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro‐inflammatory cytokine, interleukin‐1β (IL‐1β). It has not been known what this dye‐uptake path is, or whether it is involved in downstream signalling to IL‐1β release. Here, we identify pannexin‐1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye‐uptake pathway and show that signalling through pannexin‐1 is required for processing of caspase‐1 and release of mature IL‐1β induced by P2X 7 receptor activation.

/pdf/pannexin-1-mediates-large-pore-formation-and-interleukin-1b-4xozu6i4bz.pdf

Pannexin-1 mediates large pore formation and interleukin-1β release by the ATP-gated P2X7 receptor

Adenosine receptors are major targets of caffeine, the most commonly consumed drug in the world There is growing evidence that they could also be promising therapeutic targets in a wide range of conditions, including cerebral and cardiac ischaemic diseases, sleep disorders, immune and inflammatory disorders and cancer After more than three decades of medicinal chemistry research, a considerable number of selective agonists and antagonists of adenosine receptors have been discovered, and some have been clinically evaluated, although none has yet received regulatory approval However, recent advances in the understanding of the roles of the various adenosine receptor subtypes, and in the development of selective and potent ligands, as discussed in this review, have brought the goal of therapeutic application of adenosine receptor modulators considerably closer

Adenosine receptors as therapeutic targets

Saez, Juan C., Viviana M. Berthoud, Maria C. Branes, Agustin D. Martinez, and Eric C. Beyer. Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions. Physiol Rev 83: 1359-1400,...

Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions

Recently, great interest has been shown in understanding the functional roles of specific gap junction proteins (connexins) in brain, lens, retina, and elsewhere. Some progress has been made by studying knockout mice with targeted connexin deletions. For example, such studies have implicated the gap junction protein Cx36 in synchronizing rhythmic activity of neurons in several brain regions. Although knockout strategies are informative, they can be problematic, because compensatory changes sometimes occur during development. Therefore, it would be extremely useful to have pharmacological agents that block specific connexins, without major effects on other gap junctions or membrane channels. We show that mefloquine, an antimalarial drug, is one such agent. It blocked Cx36 channels, expressed in transfected N2A neuroblastoma cells, at low concentrations (IC50 ≈ 300 nM). Mefloquine also blocked channels formed by the lens gap junction protein, Cx50 (IC50 ≈ 1.1 μM). However, other gap junctions (e.g., Cx43, Cx32, and Cx26) were only affected at concentrations 10- to 100-fold higher. To further examine the utility and specificity of this compound, we characterized its effects in acute brain slices. Mefloquine, at 25 μM, blocked gap junctional coupling between interneurons in neocortical slices, with minimal nonspecific actions. At this concentration, the only major side effect was an increase in spontaneous synaptic activity. Mefloquine (25 μM) caused no significant change in evoked excitatory or inhibitory postsynaptic potentials, and intrinsic cellular properties were also mostly unaffected. Thus, mefloquine is expected to be a useful tool to study the functional roles of Cx36 and Cx50.

Potent block of Cx36 and Cx50 gap junction channels by mefloquine.

The purpose of this review is to examine the role of the extracellular A1-adenosine (Ado) receptor in modulating membrane potential and currents in cardiac cells. The cellular electrophysiological ...

Ionic basis of the electrophysiological actions of adenosine on cardiomyocytes.

How to close a gap junction channel. Efficacies and potencies of uncoupling agents.

We demonstrate that the antimalarial drug quinine specifically reduces currents through gap junctions formed by some connexins (Cx) in transfected mammalian cells, but does not affect other gap junction types. Quinine blocked Cx36 and Cx50 junctional currents in a reversible and concentration-dependent manner with half maximal blocking concentrations of 32 and 73 μM, respectively; Hill coefficients for block by quinine were about 2 for both connexins. In contrast, quinine did not substantially block gap junction channels formed by Cx26, Cx32, Cx40, and Cx43, and only moderately affected Cx45 junctions. To determine the location of the binding site of quinine (pKa = 8.7), we investigated the effect of quinine at various external and internal pH values and the effect of a permanently charged quaternary derivative of quinine. Our results indicate that the binding site for quinine is intracellular, possibly within the pore. Single-channel studies indicated that exposure to quinine induced slow transitions between open and fully closed states that decreased open probability of the channel. Quinine thus offers a potentially useful method to block certain types of gap junction channels, including those between neurons that are formed by Cx36. Moreover, quinine derivatives that are excluded from other types of membrane channels may provide molecules with connexin-specific as well as connexin-selective blocking activity.

https://www.pnas.org/content/pnas/98/19/10942.full.pdf

Quinine blocks specific gap junction channel subtypes

Aquaporin-4 (AQP4), the main water channel in the brain, is expressed in the perivascular membranes of mouse, rat, and human astrocytes. In a previous study, we used small interfering RNA (siRNA) to specifically knock down AQP4 in rat astrocyte primary cultures and found that together with reduced osmotic permeability, AQP4 knockdown (KD) led to altered cell morphology. However, a recent report on primary cultured astrocytes from AQP4 null mice (KO) showed no morphological differences compared with wild types. In this study, we compared the effect of AQP4 KD in mouse, rat, and human astrocyte primary cultures and found that AQP4 KD in human astrocytes resulted in a morphological phenotype similar to that found in rat. In contrast, AQP4 KD in mouse astrocytes caused only very mild morphological changes. The actin cytoskeleton of untreated astrocytes exhibited strong species-specific differences, with F-actin being organized in cortical bands in mouse and in stress fibers in rat and human astrocytes. Surprisingly, as a consequence of AQP4 KD, F-actin cytoskeleton was depolymerized in rat and human whereas it was completely rearranged in mouse astrocytes. Although AQP4 KD induced alterations of the cell cytoskeleton, we found that the expression of dystrophin (DP71), beta-dystroglycan, and alpha-syntrophin was not altered. AQP4 KD in cultured mouse astrocytes produced strong down-regulation of connexin43 (Cx43) with a concomitant reduction in cell coupling while no major alterations in Cx43 expression were found in rat and human cells. Taken together, these results demonstrate that with regard to these properties, human astrocytes in culture are more similar to rat than to mouse astrocytes. Moreover, even though AQP4 KD in mouse astrocytes did not result in a dramatic morphological phenotype, it induced a remarkable rearrangement of F-actin, not related to disruption of the dystrophin complex, indicating a primary role of this water channel in the cytoskeleton changes observed. Finally, the strong down-regulation of Cx43 and cell coupling in AQP4 KD mouse astrocytes indicate that a functional relationship likely exists between water channels and gap junctions in brain astrocytes.

Miduturu Srinivas

Papers

Potent block of Cx36 and Cx50 gap junction channels by mefloquine.

Ionic basis of the electrophysiological actions of adenosine on cardiomyocytes.

How to close a gap junction channel. Efficacies and potencies of uncoupling agents.

Quinine blocks specific gap junction channel subtypes

New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43