1. We have previously reported that activation of group II-like metabotropic glutamate receptors (mGluRs) in rat hippocampus results in a potentiation of the accumulation of cAMP elicited by activation of G-protein G.-coupled receptors. This large increase in cAMP levels results in release of cAMP or a cAMP metabolite and depression of synaptic transmission at the Schaffer collateral-CAl pyramidal cell synapse through activation of A1 adenosine receptors. 2. Consistent with these studies, we report that antagonists of group II mGluRs block both the potentiation of cAMP accumulation elicited by activation of mGluRs and the depression of synaptic transmission induced by coactivation of mGluRs and /J-adrenergic receptors.3. In situ hybridization studies suggest that of the cloned group II mGluRs only mGluR-3 mRNA is present in area CAl. Interestingly, mGluR-3 appears to be present predominantly in glia in this region. Thus, we tested the hypothesis that mGluRs coupled to potentiation of cAMP accumulation were present on glia rather than neurons in area CAl. 4. The selective group II mGluR agonist 2S,1'R,2'R,3'R-2-(2,3-dicarboxycyclo-propyl)glycine (DCG-IV) failed to enhance cAMP-mediated electrophysiological responses to the ,/-adrenergic receptor agonist isoprenaline (Iso) in CAl pyramidal cells, suggesting that mGluRs coupled to potentiation of cAMP accumulation may not be present in these cells. 5. Pre-incubation of hippocampal slices with either of the selective glial toxins L-a-aminoadipic acid (L-AA) or fluorocitrate (FC) blocked mGluR-mediated potentiation of cAMP accumulation. However, L-AA and FC had no discernible effects on viability of CAl pyramidal cells, or cAMP-mediated electrophysiological effects in these neurons. 6. Pre-incubation of hippocampal slices with the neurotoxin kainate resulted in disruption of neuronal transmission and degeneration of neurons in area CA1, but had no effect on mGluR-mediated potentiation of cAMP accumulation. 7. Pre-incubation of hippocampal slices with the cAMP/cAMP metabolite transport blocker probenicid blocked the depression of synaptic transmission elicited by coapplication of Iso and DCG-IV, while having no significant effect on cAMP accumulation elicited by these agonists. 8. Taken together, these data suggest that mGluRs coupled to potentiation of cAMP accumulation are present on glia rather than neurons in area CAl of hippocampus. This suggests that a novel form of glial-neuronal communication may exist, since activation of these mGluRs in concert with /J-adrenergic receptors results in depression of synaptic transmission.Glutamate, the primary excitatory neurotransmitter in the channels that mediate fast synaptic transmission. In mammalian CNS, elicits its actions by activation of both contrast, mGluRs are G-protein-coupled receptors that ionotropic and metabotropic glutamate receptors (mGluRs). regulate various second messenger systems. Since the Ionotropic glutamate receptors are ligand-gated cation discovery of the first mGluR subtype (mGluR-1) by

Malignant astrocytic gliomas, referred to as astrocytomas, represent the most commonly diagnosed adult primary brain tumor. These tumors are characterized by unrelenting growth that is often resistant to chemotherapy and radiation therapy. Tumor expansion into the healthy surrounding brain tissue produces severe and often fatal consequences. In this study, we examine the potential for the neuregulin-1/erbB receptor signaling cascade to contribute to this process by modulating glioma cell growth. Using antibodies specific for the erbB receptors, we demonstrate the expression patterns for the erbB2, erbB3, and erbB4 receptors in human glioma biopsy samples. We then verify receptor expression in a panel of human glioma cell lines. Next, we investigate the status of the erbB2 and erbB3 receptors in the human glioma cell lines and find that they are constitutively tyrosine-phosphorylated and heterodimerized. Subsequently, we demonstrate that theses same cell lines express membrane bound and released forms of neuregulins, the erbB receptor ligands, suggesting a possible autocrine or paracrine signaling network. Furthermore, we show that exogenous activation of erbB2 and erbB3 receptors in U251 glioma cells by recombinant Nrg-1beta results in enhanced glioma cell growth under conditions of serum-deprivation. This enhancement is due to an increase in cell survival rather than an increase in cell proliferation and is dependent on the activation of erbB2 and phosphatidylinositol-3 kinase (PI3K). Moreover, Nrg-1beta activates an inhibitor of apoptosis, Akt, implying a possible role for this kinase in mediating Nrg-1beta effects in gliomas. This data suggests that glioma cells may use autocrine or paracrine neuregulin-1/erbB receptor signaling to enhance cell survival under conditions where growth would otherwise be limited.

Glioma cells show up-regulation and constitutive activation of erbB2, and its expression correlates positively with increased malignancy. A similar correlation has been demonstrated for the expression of gBK, a calcium-sensitive, large-conductance K + channel. We show here that glioma BK channels are a downstream target of erbB2/neuregulin signaling. Tyrphostin AG825 was able to disrupt the constituitive erbB2 activation in a dose-dependent manner, causing a 30-mV positive shift in gBK channel activation in cell-attached patches. Conversely, maximal stimulation of erbB2 with a recombinant neuregulin (NRG-1β) caused a 12-mV shift in the opposite direction. RT-PCR studies reveal no change in the BK splice variants expressed in treated glioma cells. Furthermore, isolation of surface proteins through biotinylation did not show a change in gBK channel expression, and probing with phospho-specific antibodies showed no alteration in channel phosphorylation. However, fura-II Ca 2+ fluorescence imaging revealed a 35% decrease in the free intracellular Ca 2+ concentration after erbB2 inhibition and an increase in NRG-1β-treated cells, suggesting that the observed changes most likely were due to alterations in [Ca 2+ ] i . Consistent with this conclusion, neither tyrphostin AG825 nor NRG-1β was able to modulate gBK channels under inside-out or whole-cell recording conditions when intracellular Ca 2+ was fixed. Thus, gBK channels are a downstream target for the abundantly expressed neuregulin-1 receptor erbB2 in glioma cells. However, unlike the case in other systems, this modulation appears to occur via changes in [Ca 2+ ] i without changes in channel expression or phosphorylation. The enhanced sensitivity of gBK channels in glioma cells to small, physiological Ca 2+ changes appears to be a prerequisite for this modulation.Keywords growth factor; potassium channel; brain tumor; patch clamp; neuregulin Voltage-dependent, large-conductance, calcium-activated potassium channels (BK) differ from many other ion channels in that, as the name implies, they are activated by two physiological processes; a change in voltage and changes in intracellular Ca 2+ concentration. Under physiological conditions, Ca 2+ binding is required for channel activation. BK channels are expressed in a broad spectrum of cells and tissue types and across many species. In excitable cells, BK channel expression and activity persist throughout the life of the cell. BK channels play important roles in mediating different phases of hyperpolarization and afterhyperpolarization after an action potential and are involved in neurotransmitter release and muscle relaxation (for review see Sah, 1996;Gribkoff et al., 2001;Sah and Faber, 2002;Calderone, 2002). For nonexcitable cells, the role that these channels play in cell biology is less well understood. In glial cells, the highest channel activity is observed in undifferentiated, proliferating cells, whereas channels are largely absent in differentiated astrocytes (Puro et al., 1989;Sontheimer et al., ...