Horizontal optokinetic stimulation of rabbit retina in vivo evokes increased expression of acyl coenzyme A‐binding protein (ACBP), also known as ‘diazepam binding inhibitor,’ from retinal Müller cells. If the expressed ACBP were also secreted by Müller cells, then stimulus‐evoked secretion of ACBP could influence the activity of GABAA receptor‐expressing retinal neurons. In this study, we examine in vitro whether ACBP is secreted by Müller glial cells and Müller‐like QNR/K2 cells following stimulation with elevated levels of KCl and phorbol myristic acetate (PMA). KCl and PMA stimulation evoked secretion of threonine‐phosphorylated ACBP. A sequence analysis of ACBP shows that it has five potential phosphorylation sites: Two threonine sites fit a protein kinase C phosphorylation pattern. Two threonine sites fit a casein kinase II (CK2) pattern. One serine site fits a CK2 pattern. As CK2 is not expressed in QNR/K2 cells, it is probable that protein kinase C accounts for the phosphorylation of ACBP in these cells and for the PMA‐evoked secretion of ACBP. Serine phosphorylation was constitutive. Horizontal optokinetic stimulation increased threonine‐phosphorylated ACBP in rabbit retina. Phosphorylation of ACBP may influence its target affinity. We used a proteolytic fragment of ACBP, octadecaneuropeptide (ODN), to investigate how threonine phosphorylation influences its affinity for GABAA receptors. Threonine‐phosphorylated ODN had a stronger affinity for GABAA receptors than did unphosphorylated ODN or unphosphorylated ACBP. We conclude that stimulus‐induced Müller cell secretion of phosphorylated ACBP could influence the GABAergic transmission in neighboring retinal neurons.

Long-term horizontal optokinetic stimulation (HOKS) decreases the gain of the horizontal optokinetic reflex and evokes the second phase of optokinetic afternystagmus (OKAN-II). We investigated the possible molecular constituents of this adaptation. We used a differential display reverse transcriptase-PCR screen for mRNAs isolated from retinas of rabbits that received HOKS. In each rabbit, we compared mRNAs from the retina stimulated in the posterior3anterior (preferred) direction with mRNAs from the retina stimulated in the anterior3posterior (null) direction. Acyl-CoA-binding protein (ACBP) mRNA was one of four mRNAs selected by this screen, the proteins of which interact with GABA receptors. HOKS in the preferred direction increased ACBP mRNA transcription and ACBP protein expression. ACBP was localized to Muller glial cells by hybridization histochemistry and by immunohistochemistry. ACBP interacts with the ␣ 1 -subunit of the GABA A receptor, as determined by a yeast two-hybrid technique. This interaction was confirmed by coimmunoprecipitation of ACBP and the ␣ 1 -subunit of the GABA A receptor using an antibody to GABA A ␣ 1 . The interaction was also confirmed by a "pull-down" assay in which histidine-tagged ACBP was used to pull down the GABA A ␣ 1 . ACBP does not cross the blood-brain barrier. However, smaller truncated proteolytic fragments of ACBP do, increasing the excitability of central cortical neurons.Muller cells may secrete ACBP in the inner plexiform layer, thereby decreasing the sensitivity of GABA A receptors expressed on the surface of ganglion cell dendrites. Because retinal directional sensitivity is linked to GABAergic transmission, HOKS-induced expression of ACBP could provide a molecular basis for adaptation to HOKS and for the genesis of OKAN-II.

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.-Although ovarian surface epithelial (OSE) cells are responsible for the majority of ovarian tumors, we know relatively little about the pathway(s) that is responsible for regulating their proliferation. We found that phosphatidylinositol 3-kinase (PI3K) is activated in OSE cells in response to elevated extracellular calcium, and the PI3K inhibitors wortmannin and LY-294002 inhibited extracellular signalregulated kinase (ERK) activation by ϳ75%, similar to effects of the mitogen-activated protein kinase/ERK kinase inhibitor PD-98059. However, in assays of proliferation, we found that PD-98059 inhibited proliferation by ϳ50%, whereas wortmannin inhibited Ͼ90% of the proliferative response to elevated calcium. Expression of a dominant negative PI3K totally inhibited ERK activation in response to calcium. These results demonstrate that ERK activation cannot account for the full proliferative effect of elevated calcium in OSE cells and suggest the presence of an ERK-independent, PI3K-dependent component in the proliferative response.ovarian surface epithelial cells; signal transduction; extracellular signal-regulated kinase; mitogen-activated protein kinase; Akt; calcium-sensing receptor

Fluorescence digital imaging microscopy was used to investigate the process of myelin formation by Schwann cells in neuronal cocultures. The uptake of the fluorescent ceramide analogue N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]D-erythro-sphingosine (C5-DMB-ceramide) and its return to the plasma membrane as the corresponding fluorescent sphingomyelin and galactocerebroside analogues were measured. Through observation of this process it was possible to determine the rate of lipid synthesis in myelin internodes. The highest rate of synthesis of fluorescent sphingomyelin and galactocerebroside analogues was observed between days 3 and 7 after induction of myelination. This rate was approximately 5-fold greater than the steady-state rate of synthesis in fully myelinated internodes and 10-fold higher than the rate observed prior to myelination. The internode diameter increased during the first 3 days of myelination, but this was followed by a reduction in diameter and then an increase until the myelin sheath formation was completed. Internodes were found to be heterogeneous in terms of lipid distribution, with fluorescence intensities ranging 5-fold in myelinating cultures. Additionally, the rate of lipid transport along the internode was slow since there was a quicker increase in fluorescence intensity near the cell body of the Schwann cell than near the nodes of Ranvier. The results show that fluorescence digital imaging microscopy can be used to study the process of myelin formation and to determine the rate of formation, lipid transport, and heterogeneity of the myelin membrane.