Mechanisms of increased sensitivity for detection of thyroid stimulating antibodies by use of hypotonic medium.
We have attempted to clarify the mechanism whereby the sensitivity to detect thyroid stimulating antibodies by use of cultured thyroid cells is enhanced when sodium chloride is removed from isotonic medium. Preexposure of cultured porcine thyroid cells to 10(-4) mol/l cycloheximide increased the subsequent cAMP response to TSab stimulation in isotonic NaCl medium, but not in hypotonic NaCl-free medium. Crude membrane fractions from porcine thyroid tissues were incubated with 6 Graves' IgGs in NaCl or NaCl-free medium at 37 degrees C for 1 h, centrifuged, washed, added [125I]TSH, and incubated for another hour. The result of the 'residual TBII assay' indicated that all 6 IgGs exhibited greater inhibition of [125I]TSH binding to the membranes when pre-incubated under hypotonic than isotonic conditions. When cultured porcine thyroid cells were incubated with 7 TSab samples in NaCl or NaCl-free medium depleted with 3-isobutyl-1-methylxanthine at 37 degrees C for 1 h, washed, added the NaCl-free medium containing 3-isobutyl-1-methylxanthine, and incubated for another 2 h, the use of NaCl-free medium during the pre-incubation resulted in greater increase in cAMP elicited by all 7 samples. In summary, 1) an inhibitory mechanism upon adenylate cyclase seems to be involved under isotonic conditions and may require new protein synthesis; 2) the binding of TSH receptor antibodies to the TSH receptor increases when NaCl is removed from isotonic medium, and 3) these factors are considered responsible for the enhanced sensitivity under hypotonic conditions.
Kasagi
K
K
Department of Nuclear Medicine, Kyoto University School of Medicine, Kyoto, Japan.
Hidaka
A
A
Hatabu
H
H
Lu
C
C
Misaki
T
T
Iida
Y
Y
Konishi
J
J
D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000032:analysis; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D018828:Immunoglobulins, Thyroid-Stimulating; D012965:Sodium Chloride / Q000032:analysis; D013961:Thyroid Gland / Q000166:cytology / Q000276:immunology
D003470:Culture Media; D007074:Immunoglobulin G; D018828:Immunoglobulins, Thyroid-Stimulating; D012965:Sodium Chloride; D000242:Cyclic AMP