Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires ␣v␤5 integrin, rather than ␣v␤3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both ␣v␤3 and ␣v␤5 integrins, only ␣v␤3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of ␣v␤5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, ␣v␤5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas ␣v␤3 was expressed basolaterally. Using quantitative f luorimaging to assess in vitro uptake of f luorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, ␣v␤5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for ␣v␤5 in phagocytosis, immunof luorescence experiments demonstrated codistribution of ␣v␤5 integrin with internalized ROS. Control experiments showed that blocking ␣v␤3 function with antibodies did not inhibit ROS phagocytosis and that ␣v␤3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor ␣v␤5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-␣v␤5 complex. ␣v␤5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.Among the vital functions performed by the retinal pigment epithelium (RPE) (1) is the phagocytosis of rod outer segments (ROS) fragments (2). At birth, rat RPE cells lack phagocytic ability (3, 4). During postnatal retinal maturation, the RPE forms long, apical microvilli that ensheath developing photoreceptor outer segments. From about PN12, stacks of ROS membranes are shed daily from the distal end of photoreceptors and become efficiently phagocytosed by RPE cells (5). The essential role of RPE phagocytosis is highlighted by the rapid degeneration of photoreceptor neurons in Royal College of Surgeons rats. Royal College of Surgeons rats carry an autosomal recessive mutation that impairs RPE phagocytosis, resulting in subretinal accumulation of ROS (3, 6, 7). Photoreceptor death is irreversible and inevitably results in blindness (8, 9). RPE phagocytosis is poorly understood, compared with the well characterized phagocytosis by monocyte macrophages. RPE and systemic phagocytosis differ in that the former follows a circadian rhythm in many species (10). Furthermore, although RPE cells express Fc receptors, they highly favor ROS binding and uptake over internalization of opsonized bacteria, yeast or inert particles (11). Of special relevance to RPE phagocytosis is the phagocytosis of apoptotic cells by circulating macrophages. Clearance of senescent cells by monocyte macro...

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(βγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(βγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 μM, whereas slow mode targets neuronal NMDA receptors at around 1 μM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (

Mutations in VMD2, encoding bestrophin (best-1), cause Best vitelliform macular dystrophy (BMD), adult-onset vitelliform macular dystrophy (AVMD), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). BMD is distinguished from AVMD by a diminished electrooculogram light peak (LP) in the absence of changes in the flash electroretinogram. Although the LP is thought to be generated by best-1, we find enhanced LP luminance responsiveness with normal amplitude in Vmd2 −/− mice and no differences in cellular Cl− currents in comparison to Vmd2 +/+ littermates. The putative Ca2+ sensitivity of best-1, and our recent observation that best-1 alters the kinetics of voltage-dependent Ca2+ channels (VDCC), led us to examine the role of VDCCs in the LP. Nimodipine diminished the LP, leading us to survey VDCC β-subunit mutant mice. Lethargic mice, which harbor a loss of function mutation in the β4 subunit of VDCCs, exhibited a significant shift in LP luminance response, establishing a role for Ca2+ in LP generation. When stimulated with ATP, which increases [Ca++]I, retinal pigment epithelial cells derived from Vmd2 −/− mice exhibited a fivefold greater response than Vmd2 +/+ littermates, indicating that best-1 can suppress the rise in [Ca2+]I associated with the LP. We conclude that VDCCs regulated by a β4 subunit are required to generate the LP and that best-1 antagonizes the LP luminance response potentially via its ability to modulate VDCC function. Furthermore, we suggest that the loss of vision associated with BMD is not caused by the same pathologic process as the diminished LP, but rather is caused by as yet unidentified effects of best-1 on other cellular processes.