Intracellular calcium regulation in vagal sensory neurons

Abstract

Ca2+ regulates many cellular processes. An important mechanism for increasing cytosolic Ca2+ concentration ([Ca2+] i) is release from endoplasmic reticulum (ER) stores gated by ryanodine receptors (RyRs) and IP3 receptors (IP3Rs). The released Ca2+ can be taken up by mitochondria. Mitochondrial Ca 2+ uptake is believed to serve two functions. First, mitochondria act as a reversible store for Ca2+. Second, Ca2+ sequestration into mitochondria can regulate energy production. I have investigated the nature of the intracellular Ca2+ stores in nodose ganglion neurons (NGNs)---specifically whether the stores are functionally distinct. I have also examined the relationship between mitochondria and the intracellular Ca2+ stores. To determine if intracellular Ca2+ stores in NGNs are physiologically distinct, I used microfluorimetry, together with caffeine, a classic RyR agonist, and intracellular photorelease of caged IP3 (cIP3) to show that depletion of the RyR-gated store does not prevent Ca2+ release from the IP3R-gated store. Likewise, depletion of the IP3R-gated store does not prevent release from the RyR-gated store. Moreover, thapsigargin (TG) and cyclopiazonic acid (CPA), two inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, are able to deplete the RyR-gated Ca2+ store more effectively than the IP3R-gated Ca2+ store. These results indicate that the two Ca2+ stores are functionally distinct. Furthermore, using photorelease of cIP3 and BiNiX, a caged agonist of RyRs, I demonstrated the presence of functional IP3Rs and RyRs in the neurite extensions of cultured and acutely-dissociated NGNs. In examining mitochondria's role in Ca2+ homeostasis, I used microfluorimetry to show mitochondria can buffer increases in [Ca 2+]i caused by CICR, but not Ca 2+ influx through voltage-dependent Ca2+ channels. Simultaneous imaging of NADH/FAD+ fluorescence and intra-mitochondrial Ca2+ concentration ([Ca2+] m) revealed that mitochondrial buffering of CICR causes concomitant rises in [Ca2+]m and mitochondrial metabolism. I provide evidence that suggests a constitutive ER-to-mitochondria Ca 2+ flux. Importantly, variations of this ER-to-mitochondria Ca 2+ flux cause corresponding changes in energy metabolism, as reflected by changes in mitochondrial NADH levels. These results indicate that mitochondria are able to monitor and respond to the energy needs of the cell by buffering Ca2+ release through RyRs and by sensing the ER-to-mitochondria Ca2+ flux.