Clinical, Molecular and Biological Studies to the Genesis of Coronary Spastic Angin

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Phospholipase C(PLC)-δ1 is activated by Ca^<2+> and induces a further increment in the [Ca^<2+>]_i and enhanced response to the constrictor stimuli. We previously reported that PLC activity in the membrane fraction of the cultured skin fibroblasts obtained from the patients with coronary spastic angina is enhanced compared with that of control subjects (J Am Coll Cardiol, 2000). By the sequence analysis of the cDNA coding for PLC-δ1, we found a conversion of guanine to adenine (A) at nucleotide position 864, resulting in the amino acid replacement of arginine 257 by histidine (R257H) (Circulation, 2002). The activity of this variant PLC-δ1 protein was 2-fold higher than that of the wild-type protein. The peak increase in [Ca^<2+>] i in response to acetylcholine was greater in the cells with the variant PLC-δ1 than in those with the wild type. Thus, R257H variantin the PLC-δ1 gene can be a novel mechanism for the enhanced coronary vasomotility. However, R257H variant was detected only i … More n 10% of the patients. PLC-δ1 is negatively regulated by RhoA and positively by p122 protein. We therefore examined the possible roles of RhoA and P122 protein in the enhanced PLC-δ1 activity in CSA.Protein expression of RhoA was similar between CSA (n=5) and control patients (n=4), while that of p122 was increased in CSA (n=11) by about three-fold compared with control (n=9) (p<0.0001). Gene expression of p122 was also increased in CSA compared with control (p<0.01). Baseline intracellular calcium concentration ([Ca^<2+>]_i) and the peak increase in [Ca^<2+>]_i in response to acetylcholine were both higher in the human embryonic kidney 293 cells trans fected with p122 than in those without p122. Sequence analysis of the genomic DNA coding the promoter region of p122 (-1599 through +1) revealed 8 conversions of the nucleotide. We examined the DNA sequence in 144 CSA and 148 control patients, and one conversion of guanine to adenine at the position of -228 was more frequent in male CSA patients (8/91) than in male controls (1/62) (p<0.05). The luciferase activity in the cells transfected with the -228G/A variant promoter construct was significantly increased compared with that of wild type (p<0.001). In conclusion, p122 protein is upregulated in CSA patients, and its enhancement may be involved in the increased coronary vasomotility via the increase in [Ca^<2+>]_i. A -228G/A polymorphism is one mechanism for the upregulated p 122 protein. Less