Characterization of ryanodine receptor-mediated calcium release in human B cells: relevance to diagnostic testing for malignant hyperthermia.
BACKGROUND
Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca(2+) (Ca(2+)(i)) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH.


METHODS
Ca(2+)(i) was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca release; all experiments were performed in the absence of external Ca(2+).


RESULTS
EC(50) values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca(2+)(i) was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca(2+)(i) increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca(2+)(i) release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC(50) = 0.47 vs. 0.81 mm for cells derived from normal animals).


CONCLUSIONS
RyR1-mediated Ca(2+) signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca(2+) release from intracellular stores as an end point.
McKinney
Leslie C
LC
Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA. Lmckinney@usuhs.mil
Butler
Thomas
T
Mullen
Shawn P
SP
Klein
Michael G
MG
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D003408:Cresols / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008305:Malignant Hyperthermia / Q000175:diagnosis*; D010080:Oxazoles / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D013552:Swine
D003408:Cresols; D007093:Imidazoles; D010080:Oxazoles; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; C006984:chlorocresol; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002110:Caffeine; C061387:azumolene; D002118:Calcium