Functional Properties Of Nitric Oxide-releasable Cl- Store In Amacrine Cells

Abstract

Purpose: : We have shown that nitric Oxide (NO) releases Cl^- from an internal store in retinal amacrine cells (Hoffpauir et al. 2006). Here, we test the hypotheses that the store is sequestered and that the identity of the store could be endosomes.

Methods: : Experiments were done on cultured amacrine cells derived from 8 day chick embryos. GABA-gated currents were recorded in whole cell voltage clamp to monitor cytosolic Cl^- levels. All experiments were done with 0Cl^- pipet solution and 0Cl^- external solution to isolate the internal Cl^- store.

Results: : To confirm that NO-releasable Cl^- is sequestered, GABA_A receptor-mediated currents (50µM GABA, 400msec) were recorded from amacrine cells held at -70mV. Prior to NO, GABA-gated currents were either absent or small as residual Cl^- washed out of the cell. After NO, GABA elicited inward currents in all cells tested (n=8) indicating that sequestered Cl^- had been released. Endosomes are known to contain Cl^- and to support transmembrane proton gradients due to the activity of a V-type proton pump. To determine whether the endosomal proton gradient plays a role in the NO-dependent release of Cl^-, methylamine (10mM) was included in the pipet to buffer endosomal protons and reduce the endosomal proton gradient. With methylamine the ability of NO to release Cl^- from the store (judged by I_GABA amplitude) was significantly impaired (P=0.0006, n=17) compared to control (n=10). This is consistent with normally proton-rich endosomes being the Cl^- store. After the NO-dependent release of Cl^-, subsequent GABA responses decline as cytosolic Cl^- returns to low levels; possibly due to dialysis by the pipet, Cl^- transport mechanisms, or exit of Cl^- via activated GABA_A receptors. To reveal the contribution of GABA_A receptor-mediated Cl^- depletion, NO application was followed by 2 or 5 400 msec GABA pulses delivered over the same 3sec window. Analysis of the decline in I_GABA between the first and the final GABA pulses for both protocols demonstrated that the longer the GABAA receptors were open,the greater the decay in I_GABA(P5,66±0.07%; P2 39±0.17%; p=0.001, n=9) suggesting that under these conditions, exit via GABA_A receptors contributes substantially to the removal of cytosolic Cl^-.

Conclusions: : These results show that NO releases Cl^- from an internal compartment, possibly endosomes. Furthmore, efflux of Cl^-via GABA_A receptors plays an important role in restoring cytosolic Cl^- after a release event.

Keywords: amacrine cells • nitric oxide