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Pharmacological regulators of T cell calcium stores

Author

Alister Michael Peters, University of the Pacific

Date of Award

2004

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

David Thomas

First Committee Member

Roshanak Rahimiam

Second Committee Member

Timothy Smith

Abstract

Calcium is a crucial intracellular messenger controlling a plethora of important intracellular events. Elevated [Ca²⁺]i levels regulate numerous processes due to the versatility of the Ca²⁺ signaling in terms of speed, amplitude and spatia-temporal patterning. Depletion of intracellular calcium stores functions as a primary trigger for a message that is translated to the plasma membrane, resulting in the slow activation of plasma membrane Ca²⁺ influx channels, which allow entry of external calcium. Since these channels depend upon the state of filling of the intracellular Ca²⁺stores, these influx channels are called store-operated channels (SOCs). It is unclear how empty intracellular stores signal activation of plasma membrane capacitative calcium entry (CCE). In order to bridge the gaps in our understanding of calcium's role in T cell regulation, this project was designed to look at the effects of various pharmacological regulators of T cell calcium stores. At the start, the effort was directed at the regulation of the microsomal calcium A TPases, considering these are perhaps the most essential mediators of intracellular calcium storage. Thapsigargin (TG) and cyclopiazonic acid (CPA) were shown to be the most potent inhibitors of the Ca²⁺ -ATPase, also a new Ca²⁺ regulator aaptamine was shown to exert more modest inhibition of the Ca²⁺ pump. We also characterized a novel compound, gingerol, findings its actions are to stimulate Ca²⁺ pumps. Cell growth assays revealed an important role for ryanodine receptors (RyRs) in regulating T cell growth. RyR activators CMC and PCB95 dramatically altered T cell growth patterns leading to significantly reduced cell viability. In contrast RyR antagonist dantrolene appeared to induce growth arrest in that cell proliferation was curtailed, yet cells remained viable.

Cell viability studies revealed that the Ca²⁺ pump regulators TG and aaptamine were also observed to reduce cell growth rates, presumably as a result of their ability to deplete Ca²⁺ stores (100 nM TG was able to decrease cell viability by 90% within 24 hrs of exposure). PCB95 was able to decrease cell viability by 50% within 24 of hrs exposure and CMC decreased cell viability by 75% within 24 hrs and further over a 48 hr period. 2-APB and aaptamine were cytotoxic at higher doses. The inositol 1 ,4,5 -trisphosphate receptor (IP₃R) pathway was also found to be critically linked to T cell growth control. We observed that the IP3R modulator, 2- aminoethoxydiphenyl borate (2-APB) induced antigen-like Ca²⁺ spike that correlates with suppression of T cell growth rates.

In this study we have identified two novel T cell Ca²⁺ store regulators, aaptamine and gingerol. We also find that Ca²⁺ stores are indeed sensitively linked to T cell growth regulation. Depletion of Ca²⁺ stores with SERCA inhibitors as well as both RyR and IP₃R activators profoundly suppress T cell proliferation most likely via activation of apoptosis.

Pages

98

Recommended Citation

Peters, Alister Michael. (2004). Pharmacological regulators of T cell calcium stores. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/596