Calmodulin levels are dynamically regulated in living vascular smooth muscle cells

The total unbound calmodulin (i.e., not bound to target proteins) level in living smooth muscle cells from the ferret portal vein was monitored with a low-affinity, calmodulin-binding peptide tagged with an environmentally se nsitive fluorophore. GS17C, a previously characterized peptide, from the ca lmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2 -oxa-1,3-diazole (NBD) or, as a negative control, with iodoacetylfluorescei n isothiocyanate. Increases in NBD-GS17C fluorescence were detected by usin g confocal microscopy when chemically loaded cells were stimulated with sol utions of elevated [K+] or the calcium ionophore 4-bromoA-23187 to elicit i ncreases in intracellular Ca2+ concentration ([Ca2+](i)) quantified by fura 2. Increases in peptide fluorescence were detected in response to a phorbo l ester in the absence of changes in [Ca2+](i). These changes were blocked by the addition of the calmodulin antagonist calmidazolium. These results s uggest that the total unbound intracellular calmodulin levels may be suffic ient to regulate the activity of caldesmon and, furthermore, that phosphory lation of protein kinase C substrates may increase the level of available c almodulin in living smooth muscle cells.