The Link Between Intracellular Calcium Signaling and the Nitric Oxide-Dependent Release of Chloride in Retinal Amacrine Cells

Author

Li Zhong, Louisiana State University and Agricultural and Mechanical CollegeFollow

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Regulation of cytosolic Cl^- is an important determinant in synaptic function. Our lab has discovered that cytosolic Cl^- is regulated not only by transport across the plasma membrane but that Cl^- can also be released from internal Cl^- stores (Hoffpauir et al., 2006) in amacrine cells (ACs). Nitric oxide (NO) is a small molecule neurotransmitter that has been demonstrated to be involved in cytosolic Ca²⁺ elevations and Cl^- regulation via activation of the cystic fibrosis transmembrane regulator (CFTR) (Krishnan et al., 2017). Here, I test the hypothesis that intracellular Ca²⁺ signaling mediates the NO-dependent Cl^- regulation in retinal ACs. To determine this, I investigated the role of Ca²⁺- sensitive adenylate cyclase 1 (AdC1) in the NO-dependent release of Cl^- by applying pharmacological inhibitors and activators of cAMP pathway. Voltage-clamp recordings reveal that AdC1 plays a specific role in CFTR activation. The role of intracellular Ca²⁺ was evaluated by suppressing Ca²⁺ elevations and by artificially inducing them in zero external Ca²⁺. The results demonstrate that Ca²⁺ from internal stores is required for NO-dependent release of Cl^-. Simultaneous activation of the cAMP pathway and stimulated store release replicated the effects of NO indicating that Ca²⁺ elevations and cAMP activity are sufficient to explain the effects of NO. Insights into the mechanisms and functions of NO-dependent Ca²⁺ in cytosolic Cl^- regulation were provided by pre-applied TMEM16A inhibitor T16Ainh-A01, inositol 1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-APB and ryanodine receptor (RyR) inhibitor ryanodine, respectively in Ca²⁺ imaging and voltage- ramp recordings. T16Ainh-A01, 2-APB, and ryanodine were efficient in suppressing the NO-dependent Ca²⁺ responses and NO-dependent release of Cl^-. Co-application of T16Ainh-A01 and 2-APB produced the same inhibition in release of Cl^- as 2-APB alone, similar results were found in T16Ainh-A01 and ryanodine, indicating TMEM16A may regulate IP₃R-and RyR-related signaling upstream to the Ca2+ elevations in the NO-dependent Cl^- release. Taken together, these results suggest that AdC1 provides a link between NO-dependent cytosolic Ca²⁺ signaling and cytosolic Cl^- regulation, TMEM16A, IP₃R, and RyR are involved in the NO-dependent Ca²⁺ elevations and this signaling is necessary for cytosolic Cl^- regulation in retinal ACs.

Date

7-19-2022

Recommended Citation

Zhong, Li, "The Link Between Intracellular Calcium Signaling and the Nitric Oxide-Dependent Release of Chloride in Retinal Amacrine Cells" (2022). LSU Doctoral Dissertations. 5915.
https://repository.lsu.edu/gradschool_dissertations/5915

Committee Chair

Gleason, Evanna

DOI

10.31390/gradschool_dissertations.5915