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Nitric oxide releases Cl(-) from acidic organelles in retinal amacrine cells.

Krishnan V, Gleason E - Front Cell Neurosci (2015)

Bottom Line: Our results demonstrate that intact internal proton gradients are required for the NO-dependent release of internal Cl(-).Intriguingly, the elevation of organellar pH results in a reversal in the effects of NO.These results demonstrate that cytosolic Cl(-) is closely linked to the regulation and maintenance of organellar pH and provide evidence that acidic compartments are the target of NO.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University Baton Rouge, LA, USA.

ABSTRACT
Determining the factors regulating cytosolic Cl(-) in neurons is fundamental to our understanding of the function of GABA- and glycinergic synapses. This is because the Cl(-) distribution across the postsynaptic plasma membrane determines the sign and strength of postsynaptic voltage responses. We have previously demonstrated that nitric oxide (NO) releases Cl(-) into the cytosol from an internal compartment in both retinal amacrine cells and hippocampal neurons. Furthermore, we have shown that the increase in cytosolic Cl(-) is dependent upon a decrease in cytosolic pH. Here, our goals were to confirm the compartmental nature of the internal Cl(-) store and to test the hypothesis that Cl(-) is being released from acidic organelles (AO) such as the Golgi, endosomes or lysosomes. To achieve this, we made whole cell voltage clamp recordings from cultured chick retinal amacrine cells and used GABA-gated currents to track changes in cytosolic Cl(-). Our results demonstrate that intact internal proton gradients are required for the NO-dependent release of internal Cl(-). Furthermore, we demonstrate that increasing the pH of AO leads to release of Cl(-) into the cytosol. Intriguingly, the elevation of organellar pH results in a reversal in the effects of NO. These results demonstrate that cytosolic Cl(-) is closely linked to the regulation and maintenance of organellar pH and provide evidence that acidic compartments are the target of NO.

No MeSH data available.


Related in: MedlinePlus

Increasing compartmental pH releases Cl− into the cytosol. (A), Representative traces from three different amacrine cells recorded under zero Cl− conditions. Cells were held at −70 mV. MA and CQ labeled traces are from cells that were recorded with pipet solutions containing these compounds. Traces shown were the first collected after membrane rupture. Cartoons depict cytosolic Cl− levels under the three conditions. (B), Mean amplitudes of the first response to GABA (20 μm, 400 ms) are plotted for each of the three conditions. Both MA and CQ significantly increased the GABA-gated current amplitude over control suggesting higher internal Cl− concentration than control. (C), Leak-subtracted GABA-gated currents recorded from two different amacrine cells in 10 mM Cl− internal and external solutions. Currents were elicited by ramping the voltage from –90 mV to +50 mV in the presence of GABA (20 μM). Recordings were made just after membrane rupture. The control cell has a GABA-gated current reversal potential near the predicted equilibrium potential for Cl− of 0 mV whereas the MA-containing cell’s GABA-gated current reverses at around +15 mV. (D), Cells recorded with MA had significantly more positive reversal potentials than control. ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.
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Figure 7: Increasing compartmental pH releases Cl− into the cytosol. (A), Representative traces from three different amacrine cells recorded under zero Cl− conditions. Cells were held at −70 mV. MA and CQ labeled traces are from cells that were recorded with pipet solutions containing these compounds. Traces shown were the first collected after membrane rupture. Cartoons depict cytosolic Cl− levels under the three conditions. (B), Mean amplitudes of the first response to GABA (20 μm, 400 ms) are plotted for each of the three conditions. Both MA and CQ significantly increased the GABA-gated current amplitude over control suggesting higher internal Cl− concentration than control. (C), Leak-subtracted GABA-gated currents recorded from two different amacrine cells in 10 mM Cl− internal and external solutions. Currents were elicited by ramping the voltage from –90 mV to +50 mV in the presence of GABA (20 μM). Recordings were made just after membrane rupture. The control cell has a GABA-gated current reversal potential near the predicted equilibrium potential for Cl− of 0 mV whereas the MA-containing cell’s GABA-gated current reverses at around +15 mV. (D), Cells recorded with MA had significantly more positive reversal potentials than control. ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.

Mentions: Either 10 mM MA or 100 μm CQ was included in the pipette solution which was otherwise Cl− free. The external solution was also zero Cl−. Recordings were begun about a minute after plasma membrane rupture to allow time for the reagents to gain access to the cell. Amacrine cells were held at −70 mV and exposed to GABA pulses as in previous experiments. In control conditions, the GABA-gated currents were small and often negligible by the fifth GABA pulse. With MA internal, the pre-NO GABA-gated currents were always substantial and did not wash out over the first five GABA pulses (Figures 7A,B; mean current amplitude control 3.9 pA SD (6.1 pA); MA 79.8 pA SD (65.4 pA); n = 17; p = 0.0003). The larger currents could be entirely due to the 10 mM Cl− in the MA pipet solution (methylamine hydrochloride) or could be due to methylamine somehow stimulating release of Cl− from the internal store. If methylamine was causing release of Cl− from stores because of its ability to buffer protons in AOs, then we would expect to see a similar enhancement of GABA-gated current amplitude with CQ in the pipet.


Nitric oxide releases Cl(-) from acidic organelles in retinal amacrine cells.

Krishnan V, Gleason E - Front Cell Neurosci (2015)

Increasing compartmental pH releases Cl− into the cytosol. (A), Representative traces from three different amacrine cells recorded under zero Cl− conditions. Cells were held at −70 mV. MA and CQ labeled traces are from cells that were recorded with pipet solutions containing these compounds. Traces shown were the first collected after membrane rupture. Cartoons depict cytosolic Cl− levels under the three conditions. (B), Mean amplitudes of the first response to GABA (20 μm, 400 ms) are plotted for each of the three conditions. Both MA and CQ significantly increased the GABA-gated current amplitude over control suggesting higher internal Cl− concentration than control. (C), Leak-subtracted GABA-gated currents recorded from two different amacrine cells in 10 mM Cl− internal and external solutions. Currents were elicited by ramping the voltage from –90 mV to +50 mV in the presence of GABA (20 μM). Recordings were made just after membrane rupture. The control cell has a GABA-gated current reversal potential near the predicted equilibrium potential for Cl− of 0 mV whereas the MA-containing cell’s GABA-gated current reverses at around +15 mV. (D), Cells recorded with MA had significantly more positive reversal potentials than control. ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Increasing compartmental pH releases Cl− into the cytosol. (A), Representative traces from three different amacrine cells recorded under zero Cl− conditions. Cells were held at −70 mV. MA and CQ labeled traces are from cells that were recorded with pipet solutions containing these compounds. Traces shown were the first collected after membrane rupture. Cartoons depict cytosolic Cl− levels under the three conditions. (B), Mean amplitudes of the first response to GABA (20 μm, 400 ms) are plotted for each of the three conditions. Both MA and CQ significantly increased the GABA-gated current amplitude over control suggesting higher internal Cl− concentration than control. (C), Leak-subtracted GABA-gated currents recorded from two different amacrine cells in 10 mM Cl− internal and external solutions. Currents were elicited by ramping the voltage from –90 mV to +50 mV in the presence of GABA (20 μM). Recordings were made just after membrane rupture. The control cell has a GABA-gated current reversal potential near the predicted equilibrium potential for Cl− of 0 mV whereas the MA-containing cell’s GABA-gated current reverses at around +15 mV. (D), Cells recorded with MA had significantly more positive reversal potentials than control. ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.
Mentions: Either 10 mM MA or 100 μm CQ was included in the pipette solution which was otherwise Cl− free. The external solution was also zero Cl−. Recordings were begun about a minute after plasma membrane rupture to allow time for the reagents to gain access to the cell. Amacrine cells were held at −70 mV and exposed to GABA pulses as in previous experiments. In control conditions, the GABA-gated currents were small and often negligible by the fifth GABA pulse. With MA internal, the pre-NO GABA-gated currents were always substantial and did not wash out over the first five GABA pulses (Figures 7A,B; mean current amplitude control 3.9 pA SD (6.1 pA); MA 79.8 pA SD (65.4 pA); n = 17; p = 0.0003). The larger currents could be entirely due to the 10 mM Cl− in the MA pipet solution (methylamine hydrochloride) or could be due to methylamine somehow stimulating release of Cl− from the internal store. If methylamine was causing release of Cl− from stores because of its ability to buffer protons in AOs, then we would expect to see a similar enhancement of GABA-gated current amplitude with CQ in the pipet.

Bottom Line: Our results demonstrate that intact internal proton gradients are required for the NO-dependent release of internal Cl(-).Intriguingly, the elevation of organellar pH results in a reversal in the effects of NO.These results demonstrate that cytosolic Cl(-) is closely linked to the regulation and maintenance of organellar pH and provide evidence that acidic compartments are the target of NO.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University Baton Rouge, LA, USA.

ABSTRACT
Determining the factors regulating cytosolic Cl(-) in neurons is fundamental to our understanding of the function of GABA- and glycinergic synapses. This is because the Cl(-) distribution across the postsynaptic plasma membrane determines the sign and strength of postsynaptic voltage responses. We have previously demonstrated that nitric oxide (NO) releases Cl(-) into the cytosol from an internal compartment in both retinal amacrine cells and hippocampal neurons. Furthermore, we have shown that the increase in cytosolic Cl(-) is dependent upon a decrease in cytosolic pH. Here, our goals were to confirm the compartmental nature of the internal Cl(-) store and to test the hypothesis that Cl(-) is being released from acidic organelles (AO) such as the Golgi, endosomes or lysosomes. To achieve this, we made whole cell voltage clamp recordings from cultured chick retinal amacrine cells and used GABA-gated currents to track changes in cytosolic Cl(-). Our results demonstrate that intact internal proton gradients are required for the NO-dependent release of internal Cl(-). Furthermore, we demonstrate that increasing the pH of AO leads to release of Cl(-) into the cytosol. Intriguingly, the elevation of organellar pH results in a reversal in the effects of NO. These results demonstrate that cytosolic Cl(-) is closely linked to the regulation and maintenance of organellar pH and provide evidence that acidic compartments are the target of NO.

No MeSH data available.


Related in: MedlinePlus