The characterization of adenyl cyclase activity in FRTL-5 cell line.
The purpose of this study is to characterize the adenyl cyclase (AC) activity in Fisher rat thyroid cell line when stimulated with several agonists, such as: forskolin, sodium fluoride, Gpp(NH)p (guanosine-5'-(beta, gamma -imino) triphosphate or thyrotropin. The following studies such as: dose-response, pH, temperature, time-response, millieu concentration, 3H-thymidine uptake, Km and Vm study were also performed. Adenyl cyclase activity was measured as adenosine 3', 5'-cyclic monophosphate generated by cell cultures in 120- minutes incubation at 37 degrees C. The basal AC activity was 15.0 +/- 7.0 (mean +/- SEM, n = 6) pmol/100,000 cells/120 minutes. Forskolin at 0.1 mM increased the AC activity to 10 folds of basal AC activity. Thyroid stimulating hormone at 1 mU/ml and 10 mU/ml increased the AC activity 2 and 15 folds, respectively. Sodium fluoride stimulation study demonstrated dual actions of fluoride on adenylate cyclase; when the cells were assayed with increasing concentration of NaF, the AC activity increased as the concentration of NaF increased from 0.01 to 1 mM, but decreased strikingly as that concentration increased from 1 mM to 100 mM. When the concentration of nonhydrolyzable guanine nucleotide analogs increased in the presence of TSH, there was first an increase in adenylate cyclase activity, followed by a decrease at higher concentration. The amount of cAMP generation was much higher (P < 0.05) in hypotonic than in isotonic millieu. When the incubation temperature raised to 56 degrees C, all the AC activity was diminished. The optimal pH for AC activity was in the range of 7.4 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)
Jenq
S F
SF
Department of Laboratory Medicine, Veterans General Hospital-Taipei, Taiwan, R.O.C.
Jap
T S
TS
Hsieh
M S
MS
Chiang
H
H
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D051381:Rats; D011916:Rats, Inbred F344; D012969:Sodium Fluoride / Q000494:pharmacology; D013961:Thyroid Gland / Q000201:enzymology*; D013972:Thyrotropin / Q000494:pharmacology
D012969:Sodium Fluoride; D013972:Thyrotropin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases