Abstract
Confocal laser scanning microscopy was used to analyze alterations in nuclear free calcium (Ca2+n) levels induced by platelet-derived growth factor (PDGF) isoforms in BALB/c3T3 fibroblasts loaded with the calcium-sensitive fluorescent indicator Fluo-3. Both AA-PDGF and BB-PDGF caused a transient increase in Ca2+n. Analysis of PDGF-induced Ca2+n alterations as a function of time revealed that BB-PDGF stimulation resulted in the generation of Ca2+n oscillations that diminished over time. The frequency of BB-PDGF-stimulated oscillations was modulated by extracellular Ca2+ and could not be mimicked by increasing intracellular inositol 1,4,5-trisphosphate levels in the absence of growth factor stimulation. Caffeine alone had no effect on Ca2+n levels, but exposure of cells to caffeine after BB-PDGF stimulation augmented Ca2+n oscillations, either by increasing the frequency or reinitiating preexisting oscillations. The genesis of these oscillations in Ca2+n appears to be in the region just outside of the nucleus, as perinuclear cytoplasmic free calcium (Ca2+i) increased just prior to Ca2+n. In contrast, AA-PDGF stimulation resulted in the generation of one or two irregular, transient Ca2+n spikes. Caffeine pretreatment followed by AA-PDGF stimulation resulted in Ca2+n oscillations very similar to those produced by BB-PDGF alone. Additionally, the AA-PDGF and BB-PDGF isoforms appeared to modulate distinct pools of cellular Ca2+, as BB-PDGF was still capable of inducing Ca2+n oscillations subsequent to prior induction of oscillations by AA-PDGF/caffeine. These PDGF isoform-specific changes in nuclear free Ca2+ could serve as a mechanism by which isoform-specific cellular signaling pathways may be manifested by the growth factors.
Highlights
Confocal laser scanning microscopy was used toana- the existence of three PDGF isofonns that possess the capacity lyze alterationsin nuclear free calcium (Ca2+,) levels itno-interact differentially with at least two distinct but related duced by platelet-derivedgrowthfactor (PDGF)iso- receptors [2].PDGF molecules exist as homodimers (AA-and formsin BALB/&TSfibroblasts loaded wtihthe calcium- BB-PDGF)or heterodimers ( ~ - P D G Fo)f two disulfide-linked sensitive fluorescent indicator Fluo-3
Lation augmentedCa", oscillations,either by increasing the frequency or reinitiating preexisting oscillations. The genesisof these oscillations Cinasn appearsto be in the region just outside of the nucleus, as perinuclear the PDGF B chain with high affinity
AA-PDGF stimulation resulted inthe ligands, one of the early cellular events initiated by PDGF is a generation of one or two irregular, transient Ca2+, rapid, transient elevation in cytoplasmic calcium(Ca2+,()6, 7)
Summary
Cell Culture-BALBlc murine 3T3fibroblasts(cloneA31) were grown as described [8, 16].Cellswere maintained in Dulbecco'smodified. Cells weremade quiescent prior to PDGF stimulation by placement in serum-free medium (Dulbecco's modified Eagle's medium/Ham's F-12, 1:1, supplemented with insulin/transferrin/selenium) 48 h before use. Uiescent BALB/c3T3 cells were loaded with Fluo-3/Ahl,and images from an optical section through the center of the nucleus were obtained byconfocal laser scanning microscopy at 5-s intervals. Represented on they axis is the average fluorescent pixel intensity of the mapped area of a single nucleus. Cells cultured on glass coverslips were loadedwith Caz+-sensitive fluorophore (see below), and imaged by collecting a single 768 x 512 pixel scan from the same focal plane once every 5-15 s using the diluted to 500 ng/ml in Dulbecco's modified Eagle'smedium/Ham's F-12 medium containing 0.1%bovine serum albumin and neutralized with 5 mM Na,CO,. Rapid confocaline scanning was performed using the Olympus LS&i
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