Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultaneously. Moreover, imaging Ca transients using Ca-sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive genetically-encoded Ca indicators (GECIs) it is now possible to observe changes in Ca transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock-in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia (DRG), spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism's biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential methodological considerations.
Afferent Pathways ; physiology ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; genetics ; Ganglia, Spinal ; metabolism ; Humans ; Pain ; metabolism ; pathology

PURPOSE: The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca(2+) signaling. However, whether the changes in Ca(2+) signaling and Ca(2+) signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2(+/-) mouse parotid gland acinar cells, Ca(2+) signaling, expression levels of Ca(2+) signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2(+/-) mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca(2+) ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP(3)Rs), but the localization and activities of IP3Rs were not altered. In SERCA2(+/-) mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca(2+) signaling proteins in the parotid gland acini, however, overall Ca(2+) signaling is unchanged.
Amylases/metabolism ; Animals ; Blotting, Western ; Calcium/metabolism ; Calcium Signaling/drug effects/genetics/*physiology ; Carbachol/pharmacology ; Immunohistochemistry ; Inositol 1,4,5-Trisphosphate Receptors/metabolism ; Mice ; Mice, Knockout ; Parotid Gland/*metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics/*metabolism ; Signal Transduction/drug effects/genetics/physiology

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

OBJECTIVESTo investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis.

METHODSFree intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells.

RESULTSThere were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment.

CONCLUSIONSThe high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).

Calcium ; metabolism ; Calcium Signaling ; drug effects ; Carcinoma, Giant Cell ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; genetics ; Humans ; Lung Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Receptor, PAR-1 ; genetics ; metabolism ; physiology ; Receptors, Thrombin ; metabolism ; Thrombin ; pharmacology ; Transfection ; Up-Regulation