DIFFERENTIAL MUSCARINIC RECEPTOR MESSENGER-RNA EXPRESSION BY FRESHLY ISOLATED AND CULTURED BOVINE AORTIC ENDOTHELIAL-CELLS

Endothelial cells, either in vivo or freshly isolated, respond when exposed to muscarinic agonists with an increase in cytosolic free calcium concentration ([Ca2+]i) and release of endothelium-derived relaxing factor (EDRF). When placed in culture, however, endothelial cells rapidly lose these responses, which may be related to changes in muscarinic receptor expression. Northern blot analysis of poly(A)+ RNA from freshly isolated or cultured bovine aortic endothelial cells was used to address this problem. Through the use of specific cDNA probes complementary to the nonconserved regions of the m1, m2, m3, m4, and m5 muscarinic receptors, mRNA transcripts for the m1 (3.9 kb), m2 (3.8 kb), and m3 (3.1 kb) receptor subtypes were identified in freshly isolated endothelial cells, whereas ml and m3 transcripts were identified in aortic smooth muscle. In contrast, cultured endothelial cells contained mRNA for only the m2 receptor subtype. Transcripts for the m4 or m5 receptors were not detected in either freshly isolated or cultured endothelial cells. Since ml and m3 receptor subtypes are coupled to phospholipase C, activation of which is required for EDRF release, these observations may explain the failure of muscarinic agonists to elicit a rise in [Ca2+]i and EDRF release from cultured endothelial cells.