Long-lasting pain may partly be a consequence of ongoing neuroinflammation, in which astrocytes play a significant role. Following noxious stimuli, increased inflammatory receptor activity, influences in Na(+)/K(+)-ATPase activity and actin filament organization occur within the central nervous system. In astrocytes, the Ca(2+) signaling system, Na(+) transporters, cytoskeleton, and release of pro-inflammatory cytokines change during inflammation. The aim of this study was to restore these cell parameters in inflammation-reactive astrocytes. We found that the combination of (1) endomorphin-1, an opioid agonist that stimulates the Gi/o protein of the μ-opioid receptor; (2) naloxone, an opioid antagonist that inhibits the Gs protein of the μ-opioid receptor at ultralow concentrations; and (3) levetiracetam, an anti-epileptic agent that counteracts the release of IL-1β, managed to activate the Gi/o protein and Na(+)/K(+)-ATPase activity, inhibit the Gs protein, and decrease the release of IL-1β. The cell functions of astrocytes in an inflammatory state were virtually restored to their normal non-inflammatory state and it could be of clinical significance and may be useful for the treatment of long-term pain.

Connexin43 (cx43) forms gap junctions in astrocytes, and these gap junctions mediate intercellular communication by providing transport of low-molecular-weight metabolites and ions. We have recently shown that systemic growth hormone increases cx43 in the brain. One possibility was that local brain insulin-like growth factor-I (IGF-I) could mediate the effect by acting directly on astrocytes. In the present study, we examined the effects of direct application of recombinant human IGF-I (rhIGF-I) on astrocytes in primary culture concerning cx43 protein expression and gap junctional communication (GJC). After 24 hr of stimulation with rhIGF-I under serum-free conditions, the GJC and cx43 protein were analyzed. Administration of 30 ng/ml rhIGF-I increased the GJC and the abundance of cx43 protein. Cell proliferation of the astrocytes was not significantly increased by rhIGF-I at this concentration. However, a higher concentration of rhIGF-I (150 ng/ml) had no effect on GJC/cx43 but increased cell proliferation. Because of the important modulatory role of IGF binding proteins (IGFBPs) on IGF-I action, we analyzed IGFBPs in conditioned media. In cultures with a low abundance of IGFBPs (especially IGFBP-2), the GJC response to 30 ng/ml rhIGF-I was 81%, compared with the average of 25%. Finally, as a control, insulin was given in equimolar concentrations. However, GJC was not affected, which suggests that rhIGF-I acted via IGF-I receptors. In summary, the data show that rhIGF-I may increase GJC/cx43, whereas a higher concentration of rhIGF-I--at which stimulation of proliferation occurred--did not affect GJC/cx43. Furthermore, IGFBP-2 appeared to modulate the action of rhIGF-I on GJC in astrocytes by a paracrine mechanism.

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Astrocytic responses to activation of metabotropic glutamate receptors group I (mGluRs I) and ␣ 1 -adrenoreceptors in cultured cells have been assessed using spectral analyzes and calcium imaging. Concentrationdependent changes were observed after stimulation with the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG). These responses changed from a regular low frequency signal with sharp peaks at 1 M to a pronounced stage of irregularity at 10 M. After stimulation with 100 M the signal was again homogenous in shape and regularity but occurred at a higher frequency. In contrast, the spectral properties after stimulation with the ␣ 1 -adrenoreceptor agonist phenylephrine, exhibited considerable variation for all investigated concentrations. DHPG-induced increases in [Ca 2؉ ] i were also associated with astroglial glutamate release, whereas no release was observed after noradrenergic stimulation. Both DHPG-mediated calcium signaling and glutamate release were inhibited by preincubation with 10 or 100 M phenylephrine. Collectively, the present investigation provides new information about the spatialtemporal characteristics of astroglial intracellular calcium responses and demonstrates distinct differences between noradrenergic and glutamatergic receptors regarding intracellular calcium signaling and coupling to glutamate release. The noradrenergic modulation of DHPG-induced responses indicates that intracellular astroglial processes can be regulated in a bi-directional feedback loop between closely connected astrocytes and neurons in the central nervous system. Astrocytes, the main population of glial cells, form gap junction-coupled multicellular networks and communicate among themselves through variations in intracellular calcium concentration ([Ca 2ϩ ] i ) 1 (1). These cells express a calcium-based excitability in which receptor-mediated calcium elevations can propagate as intracellular waves over considerable distances (1, 2). A diverse range of [Ca 2ϩ ] i changes are induced in astrocytes after exposure to various neurotransmitters (2-6) and the cells display [Ca 2ϩ ] i oscillations and intracellular waves in response to neuronal activity (7-9). Previous results demonstrate the importance of glutamate for the mediation of these neuronal-glial interactions. However, the potential contribution of other important neurotransmitters such as those belonging to the noradrenergic system has not yet been fully investigated.Variations in astrocytic [Ca 2ϩ ] i mediate the uptake and release of different neuroactive substances (10) such as arachidonic acid, neurotrophins, and neurotransmitters, and via this mechanism possibly modulate synaptic transmission (11-13). Consistent with this, previous studies have shown that activation of glial metabotropic glutamate receptors in concert with ␤-adrenoreceptors (␤-AdRs) can result in depression of synaptic activity in which the release of cAMP from astrocytes is central (14). Furthermore, adrenergic and glutamatergic interactions can also greatly affect both cAMP-and ...

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