Abstract
We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses.
Highlights
Cells have two major mechanisms available for regulating the release of internal Ca2ϩ
The intron identified within the human RyR3 gene is in the same position as an intron in the pig RyR1 gene, suggesting that some intron splice sites may be conserved between ryanodine receptors (RyRs) isoforms and animal species
Of particular interest were the myogenic cell lines BC3H1, Sol8, C2C12, and L6, where the intensity of the 530-bp PCR product correlated with the differentiation of the cells from a non-muscle to a muscle phenotype
Summary
Cells have two major mechanisms available for regulating the release of internal Ca2ϩ. Evidence for the expression of RyRs in various excitable and nonexcitable tissues has mostly been obtained using either molecular or pharmacological methods. These approaches have demonstrated the tissue-specific expression and function of RyRs For example, effects of ryanodine on agonistinduced calcium signals in intact cells [9, 10] and specific ryanodine binding sites in hepatocyte vesicles [11, 12] have been demonstrated. For other nonexcitable cells the converse problem exists, in that molecular techniques have identified RyR mRNA expression, but the functional evidence has been confusing, since some RyR-activating agents fail to evoke responses in these cells. In mink lung epithelial cells and Jurkat T-lymphocytes, which appear to express RyR3, effects of ryanodine but not caffeine have been observed [3, 16]. In a separate study Guse et al [17] found that Jurkat cells were caffeine-responsive
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