Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor.
Previous studies have demonstrated a gain-of-function of the calcium-sensing receptor (CASR) gene R990G polymorphism. In this study, activation of the R990G CASR stably transfected in HEK-293 (HEK-990G) cells compared with that of the common variant (HEK-wild-type (WT)) by increasing concentrations of CaCl(2) or calcimimetic R-568 caused significantly higher intracellular free calcium concentration ([Ca(2+)](i)) and lower Ca-EC(50). Moreover, the [Ca(2+)](i) oscillation percentage was higher with a larger sinusoidal pattern in HEK-990G. R-568 induced a shift of the oscillatory events from 4 to 2  mmol/l extracellular calcium concentration in HEK-990G cells and increased the sinusoidal oscillation percentage in comparison with HEK-WT. Preincubation with thapsigargin or phospholipase C inhibitors completely prevented oscillations in both cell lines, consistent with the involvement of the inositol trisphosphate pathway, while protein kinase C inhibitor prevented oscillations in HEK-WT cells only. Finally, CaCl(2) and R-568 caused a significant increase in p44/42 extracellular signaling-regulated kinase phosphorylation, with the mean Ca-EC(50) values being significantly lower in HEK-990G. Our findings demonstrated that the 990G allele is associated with high sensitivity to R-568, which provided new evidence for differences in CASR signaling.
Terranegra
Annalisa
A
Department of Medicine Surgery and Dentistry, IRCCS Policlinico San Donato, Milan, Italy. annalisa.terranegra@unimi.it
Ferraretto
Anita
A
Dogliotti
Elena
E
Scarpellini
Milena
M
Corbetta
Sabrina
S
Barbieri
Anna Maria
AM
Spada
Anna
A
Arcidiacono
Teresa
T
Rainone
Francesco
F
Aloia
Andrea
A
Cusi
Daniele
D
Vezzoli
Giuseppe
G
Soldati
Laura
L
D019943:Amino Acid Substitution / Q000235:genetics*; D000814:Aniline Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010627:Phenethylamines; D010766:Phosphorylation / Q000187:drug effects; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011437:Propylamines; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D044169:Receptors, Calcium-Sensing / Q000235:genetics*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000814:Aniline Compounds; D020536:Enzyme Activators; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D047428:Protein Kinase Inhibitors; D044169:Receptors, Calcium-Sensing; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D010738:Type C Phospholipases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium