Abstract-Despite intensive interest in the dedifferentiation process of vascular smooth muscle cells, very little data are available on intracellular Ca 2ϩ signaling. The present study was designed to investigate the evolution of the intracellular Ca 2ϩ pools when rat aortic smooth muscle cells (RASMCs) proliferate and to define the mechanisms involved in the functional alterations. RASMCs were cultured in different conditions, and [Ca 2ϩ ] i was measured by use of fura 2. Expression of the sarco(endo)plasmic reticulum Ca 2ϩ pumps (SERCA2a and SERCA2b), Ca 2ϩ channels, the ryanodine receptor (RyR), and the inositol trisphosphate receptor (IP3R) was studied by reverse transcription-polymerase chain reaction and immunofluorescence. Antibodies specific for myosin heavy chain isoforms were used as indicators of the differentiation state of the cell, whereas an anti-proliferating cell nuclear antigen antibody was a marker of proliferation. SERCA2a, SERCA2b, RyR3, and IP3R-1 mainly were present in the aorta in situ and in freshly isolated RASMCs. These cells used the 2 types of Ca 2ϩ channels to release Ca 2ϩ from a common thapsigargin-sensitive store. Proliferation of RASMCs, induced by serum or by platelet-derived growth factor-BB, resulted in the disappearance of RyR and SERCA2a mRNAs and proteins and in the loss of the caffeine-and ryanodine-sensitive pool. The differentiated nonproliferative phenotype was maintained in low serum or in cells cultured at high density. In these conditions, RyR and SERCA2a were also present in RASMCs. Thus, expression of RyR and SERCA2a is repressed by cell proliferation, inducing loss of the corresponding Ca 2ϩ pool. In arterial smooth muscle, Ca 2ϩ release through RyRs is involved in vasodilation, and suppression of the ryanodine-sensitive pool might thus alter the control of vascular tone.

Background-The response of ventricular myocytes to pressure overload is heterogeneous and not spatially coordinated.We investigated whether or not the alterations in SERCA and RyR gene expression are homogeneous within the myocardium. Methods and Results-The cellular distribution of mRNAs and proteins encoding the 2 sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) isoforms (SERCA 2a and 2b) and 2 Ca 2ϩ release channels (the ryanodine receptor, RyR, and the IP 3 receptor, IP 3 R) were analyzed by in situ hybridization and immunofluorescence, respectively. Analyses were performed during early (1 and 5 days) and late (1 month) stages of cardiac hypertrophy induced in rat by thoracic aortic stenosis (AS). The results indicated that 1 and 5 days after AS, the cellular distribution of SERCA 2a and RyR2 mRNAs in right ventricle and atrium was similar to controls but the mRNA levels appeared to decrease in some areas of the left ventricle (LV). One month after AS, the distribution of SERCA 2a mRNA and protein became heterogeneous throughout the LV, whereas RyR2 mRNA and protein levels were decreased in a homogeneous manner. SERCA 2b, poorly expressed in both cardiomyocytes and vessels of controls, was increased 4-fold 1 month after AS in coronary arteries only. In both sham (Sh) and AS, SERCA 3 and IP 3 R mRNAs were mainly found in the vessels. Conclusions-In