Regulation of calcium release from the endoplasmic reticulum in dorsal root ganglia neurons

Ca2+ is an important second messenger in numerous cellular signaling pathways, necessitating tight control of intracellular Ca 2+ ([Ca2+]i) homeostasis. The endoplasmic reticulum (ER) plays an important role in controlling [Ca2+] i, serving as both source and sink for Ca2+ signals. Studying pathways that control Ca2+ homeostasis in sensory neurons provides insight into the role of Ca2+ signaling in sensory physiology. The objective of these studies was to determine mechanisms regulating Ca2+ release from the ER and to identify functional consequences of these signals in rat sensory neurons.; Ca2+-induced Ca2+-release (CICR) via ryanodine receptors on the ER membrane can amplify transient elevations in [Ca 2+]i. In the initial study presented here, we described regenerative CICR and [Ca2+]i oscillations evoked in a population of rat dorsal root ganglion neurons upon coincident exposure to caffeine and depolarization. CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane. We describe a tunable threshold for regenerative CICR that may be modulated by agents that alter the sensitivity of the ryanodine receptor. Mitochondria regulated CICR by providing ATP and buffering [Ca2+] i. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca 2+ pump were affected by inhibition of aerobic metabolism. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.; Changes in the sensory characteristics of dorsal root ganglion neurons are a common consequence of trauma and inflammation. The mechanisms leading to sustained sensitization are still poorly understood, but are hypothesized to depend on alogenic agents released following tissue injury. Bradykinin produced at sites of tissue injury and inflammation, elicits acute pain and alters the sensitivity of nociceptive neurons to subsequent stimuli. We tested the hypothesis that bradykinin could elicit long lasting changes in nociceptor function by activating members of the Nuclear Factor of Activated T-cells (NFAT) family of transcription factors. Bradykinin activation of B2 receptors evoked concentration dependent increases [Ca2+] i in a proportion of dorsal root ganglion neurons in primary culture. These [Ca2+] increases depended on phospholipase C (PLC) and Ca2+ store mobilization. In neurons expressing a green fluorescent protein (GFP)-NFAT4 fusion protein, exposure to bradykinin induced the translocation of GFP-NFAT4 from the cytoplasm to the nucleus. Translocation was partially inhibited by removal of extracellular Ca2+ and was blocked by inhibition of calcineurin. Furthermore, bradykinin triggered a concentration-dependent increase in NFAT-mediated transcription of a luciferase gene reporter. This depended on the B2 receptor, PLC activation and IP3 mediated Ca2+ release. Transcription was not inhibited by capsazepine. Lastly, as indicated by quantitative RT-PCR, bradykinin elicited an increase in cyclooxygenase (cox-2) mRNA. This increase was sensitive to calcineurin and B2 receptor inhibition. These findings suggest a mechanism by which short-lived bradykinin-mediated stimuli can enact lasting changes in nociceptor function and sensitivity.; In summary, I describe two aspects of ER Ca2+ signaling that operate in sensory neurons. These investigations demonstrate novel regulation of a Ca2+ release pathway by mitochondria and delineate a pathway linking Ca2+ release to long-term changes in sensory neuron function. In the first study, a novel mechanism coupling aerobic metabolism to ER Ca2+ signaling was identified. This study provides insight into the interconnect...