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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.


The NO-dependent Cl− store is sequestered. (A), Representative traces from an amacrine cell recorded in the whole-cell voltage clamp configuration with Cl− free pipette and external solutions. The cell was held at −70 mV and pulses of GABA (20 μM, 400 ms) were applied. Injection of NO temporarily increased the amplitude of the GABA-gated currents indicating a release of Cl− into the cytosol. Cartoons at the top depict the Cl− distribution at the point in time being sampled directly below. The darker color represents more Cl−. (B), Current amplitude data are plotted for each cell recorded. Data were collected from the response to the first GABA pulse and from the response to the 6th GABA pulse delivered just after NO. (C), Mean current amplitude of the data shown in (B). (D,E), Data from an experiment like the one shown in (A) but here the NO donor NOC-5 (500 μM) is used rather than the NO-bubbled solution. The NO donor also causes a significant increase in the amplitude in the NO-dependent GABA-gated currents. (F), Mean NO-dependent current amplitude of GABA-gated currents recorded before and after the addition of acetazolamide (400 μm). **** denotes p < 0.0001.
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Figure 2: The NO-dependent Cl− store is sequestered. (A), Representative traces from an amacrine cell recorded in the whole-cell voltage clamp configuration with Cl− free pipette and external solutions. The cell was held at −70 mV and pulses of GABA (20 μM, 400 ms) were applied. Injection of NO temporarily increased the amplitude of the GABA-gated currents indicating a release of Cl− into the cytosol. Cartoons at the top depict the Cl− distribution at the point in time being sampled directly below. The darker color represents more Cl−. (B), Current amplitude data are plotted for each cell recorded. Data were collected from the response to the first GABA pulse and from the response to the 6th GABA pulse delivered just after NO. (C), Mean current amplitude of the data shown in (B). (D,E), Data from an experiment like the one shown in (A) but here the NO donor NOC-5 (500 μM) is used rather than the NO-bubbled solution. The NO donor also causes a significant increase in the amplitude in the NO-dependent GABA-gated currents. (F), Mean NO-dependent current amplitude of GABA-gated currents recorded before and after the addition of acetazolamide (400 μm). **** denotes p < 0.0001.

Mentions: Decay indices (Figure 3) were calculated by the following formulae: DI = 1 − (amp P5/amp P1) or DI = 1 − (amp P2/amp P1) with “amp” indicating GABA-gated current amplitude. Data were analyzed using Origin 8.0 (OriginLab, Northampton, MA) analysis software and data are presented as means ± SD. Data were generally evaluated using the paired and unpaired student’s t-test as appropriate. Levels of significance are denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


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

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

The NO-dependent Cl− store is sequestered. (A), Representative traces from an amacrine cell recorded in the whole-cell voltage clamp configuration with Cl− free pipette and external solutions. The cell was held at −70 mV and pulses of GABA (20 μM, 400 ms) were applied. Injection of NO temporarily increased the amplitude of the GABA-gated currents indicating a release of Cl− into the cytosol. Cartoons at the top depict the Cl− distribution at the point in time being sampled directly below. The darker color represents more Cl−. (B), Current amplitude data are plotted for each cell recorded. Data were collected from the response to the first GABA pulse and from the response to the 6th GABA pulse delivered just after NO. (C), Mean current amplitude of the data shown in (B). (D,E), Data from an experiment like the one shown in (A) but here the NO donor NOC-5 (500 μM) is used rather than the NO-bubbled solution. The NO donor also causes a significant increase in the amplitude in the NO-dependent GABA-gated currents. (F), Mean NO-dependent current amplitude of GABA-gated currents recorded before and after the addition of acetazolamide (400 μm). **** denotes p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4459082&req=5

Figure 2: The NO-dependent Cl− store is sequestered. (A), Representative traces from an amacrine cell recorded in the whole-cell voltage clamp configuration with Cl− free pipette and external solutions. The cell was held at −70 mV and pulses of GABA (20 μM, 400 ms) were applied. Injection of NO temporarily increased the amplitude of the GABA-gated currents indicating a release of Cl− into the cytosol. Cartoons at the top depict the Cl− distribution at the point in time being sampled directly below. The darker color represents more Cl−. (B), Current amplitude data are plotted for each cell recorded. Data were collected from the response to the first GABA pulse and from the response to the 6th GABA pulse delivered just after NO. (C), Mean current amplitude of the data shown in (B). (D,E), Data from an experiment like the one shown in (A) but here the NO donor NOC-5 (500 μM) is used rather than the NO-bubbled solution. The NO donor also causes a significant increase in the amplitude in the NO-dependent GABA-gated currents. (F), Mean NO-dependent current amplitude of GABA-gated currents recorded before and after the addition of acetazolamide (400 μm). **** denotes p < 0.0001.
Mentions: Decay indices (Figure 3) were calculated by the following formulae: DI = 1 − (amp P5/amp P1) or DI = 1 − (amp P2/amp P1) with “amp” indicating GABA-gated current amplitude. Data were analyzed using Origin 8.0 (OriginLab, Northampton, MA) analysis software and data are presented as means ± SD. Data were generally evaluated using the paired and unpaired student’s t-test as appropriate. Levels of significance are denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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.