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 sensitive fluorophore. GS17C, a previously characterized peptide, from the calmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2-oxa-1,3-diazole (NBD) or, as a negative control, with iodoacetylfluorescein isothiocyanate. Increases in NBD-GS17C fluorescence were detected by using confocal microscopy when chemically loaded cells were stimulated with solutions of elevated [K(+)] or the calcium ionophore 4-bromoA-23187 to elicit increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) quantified by fura 2. Increases in peptide fluorescence were detected in response to a phorbol ester in the absence of changes in [Ca(2+)](i). These changes were blocked by the addition of the calmodulin antagonist calmidazolium. These results suggest that the total unbound intracellular calmodulin levels may be sufficient to regulate the activity of caldesmon and, furthermore, that phosphorylation of protein kinase C substrates may increase the level of available calmodulin in living smooth muscle cells.
Hulvershorn
J
J
Signal Transduction Group, Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Gallant
C
C
Wang
C A
CA
Dessy
C
C
Morgan
K G
KG
D009327:4-Chloro-7-nitrobenzofurazan; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D005289:Ferrets; D007202:Indicators and Reagents; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D011169:Portal Vein / Q000166:cytology / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007202:Indicators and Reagents; D011494:Protein Kinases; D009327:4-Chloro-7-nitrobenzofurazan; D002118:Calcium