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SA Keirstead, JM Reifel Saltzberg; Calcium Waves in Isolated Müller Cells Do Not Require an Internally Propagated Signal . Invest. Ophthalmol. Vis. Sci. 2002;43(13):671.
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Purpose: Purinergic agonists evoke a wave-like increase in intracellular calcium ion concentration ([Ca2+]i) in isolated tiger salamander Müller cells This response occurs first in the distal/soma region and later in the endfoot. The purpose of this study was to determine whether these calcium waves are mediated by an internally propagated signal or if they are due to some inherent delay in the response in the endfoot region. Methods: Müller cells were enzymatically and mechanically dissociated from retinas of larval tiger salamanders and loaded with the calcium-sensitive dye Fura-2 AM. Cells were plated onto a coverslip-bottomed perfusion chamber which was placed on the stage of an inverted fluorescence microscope equipped with a computer-controlled filter wheel and digital camera. Fluorescence ratioes were measured in selected regions and plotted versus time. ATP (10-100 microM), 2-methylthio ATP (2MeSATP; 0.5 microM) and MRS2179 (500 microM) were applied either to the bath or locally by pressure ejection with a micropipette. Tetramethyl rhodamine (0.35%) was included in the pipette to monitor the distribution of the ejected solution. Results: Local application of ATP to either the distal region or endfoot of single Müller cells resulted in an increase in ([Ca2+]i) in the region to which ATP was applied. Thus, responses in either the distal region or endfoot region were localized and were not propagated to other regions of the cells. Localized application of the P2Y1 receptor antagonist MRS2179 to the endfoot of Müller cells inhibited responses to bath-applied 2MeSATP in the endfoot while the distal and somatic region of the cells responded as in the control situation. When the shaft connecting the soma and endfoot of Müller cells was transected, the endfoot retained the ability to respond to bath-applied ATP and the cells exhibited wave-like [Ca2+]i increases similar to those in intact cells. Conclusion: Wave-like [Ca2+]i responses in isolated Müller cells are not mediated by an internally propagated signal but rather the endfoot is capable of responding to purinergic agonists independent of the rest of the cell. These data suggest that the endfoot responses to purinergic agonists are inherently delayed with respect to distal/somatic responses. The mechanism for this delay is not yet clear, but these data indicate that the endfeet of Müller cells could respond to ATP released by amacrine cells in the inner retina.
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