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Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell [Cl.sup.-] currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[[sup.3]H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[[sup.3]H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked ([greater than or equal to] 80% inhibition) by intracellular [Ca.sup.2+] chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[[sup.3]H]aspartate release activated by the substantial hyposmotic swelling showed little ([less than or equal to] 25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate [Ca.sup.2+]-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate [Ca.sup.2+]/calmodulin-independent signaling mechanism. [Ca.sup.2+]-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 [micro]M) activated a small EAA release in 70-80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5-10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo. cell swelling; anion channels; glutamate release; adenosine 5'-triphosphate release; neuron-glia communication

A study was conducted to analyze the involvement of tyrosine phosphorylation in hyposmotic medium-induced (3H)taurine and D-(3H)aspartate release from primary astrocyte cultures. Results indicated that the release of 3-(3H)aspartate was not affected by agents in the same concentration range. Findings also showed that the inactive analog tyrphostin 1 had no influence on the release of amino acids.

Research was conducted to characterize the swelling-activated channel(s) responsible for D-(3H)aspartate release from primary cultured astrocytes exposed to either hypotonic medium or KCl during stroke or head trauma. Results indicate that a volume-sensitive channel causes D-(3H)aspartate release from primary cultured astrocytes during hypotonic medium-induced and raised extracellular K+ release.

Volume changes in monolayer cell cultures attached to asubstratum can be measured continuously by using the electrical resistance of the medium as a monitor of the amount of fluid absorbed or extruded from the cells. The culture is suspended in a balanced salt solution and confined in a channel where the electrical resistance can be measured. Resistance is inversely proportional to the amount of water absorbed by the cell and is used as a gauge of the volume changes. The method can measure cell volume changes with a range of 1% to 2%. Measurement of the volume changes in an astrocyte culture is presented as an example.

Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

Mongin, Alexander A., Zhaohui Cai, and Harold K. Kimelberg. Volume-dependent taurine release from cultured astrocytes requires permissive [[Ca.sup.2+]]i and calmodulin. Am. J. Physiol. 277 (Cell Physiol. 46): C823-C832, 1999.--Cell swelling results in regulatory activation of multiple conductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement of extracellular and intracellular [Ca.sup.2+] in volume-dependent [[sup.3]H]taurine efflux from primary cultured astrocytes and compare the [Ca.sup.2+] sensitivity of this efflux in slow (high [K.sup.+] medium induced) and fast (hyposmotic medium induced) cell swelling. Neither [Ca.sup.2+]-free medium nor [Ca.sup.2+]-channel blockers prevented the volume-dependent [[sup.3]H]taurine release. In contrast, loading cells with the membrane-permeable [Ca.sup.2+] chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed [[sup.3]H]taurine efflux by 65-70% and 25-30% under high-[K.sup.+] and hyposmotic conditions, respectively. Fura 2 measurements confirmed that BAPTA-AM, but not [Ca.sup.2+]-free media, significantly reduced resting intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]]i). The calmodulin antagonists trifluoperazine and fluphenazine reversibly and irreversibly, respectively, inhibited the high-[K.sup.+]-induced [[sup.3]H]taurine release, consistent with their known actions on calmodulin. In hyposmotic conditions, the effects were less pronounced. These data suggest that volume-dependent taurine release requires minimal basal [[Ca.sup.2+]]i and involves calmodulin-dependent step(s). Quantitative differences in [Ca.sup.2+]/calmodulin sensitivity of high-[K.sup.+]. induced and hyposmotic medium-induced taurine efflux are due to both the effects of the inhibitors on high-[K.sup.+]-induced cell swelling and their effects on transport systems and/or signaling mechanisms determining taurine efflux. cell swelling; brain edema; anion channels; calcium dependence; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; trifluoperazine; intracellular calcium concentration