Pdgf-BB Triggered Cytoplasmic Calcium Responses in Cells with Endogenous or Stably Transfected PDGF β-Receptors

Abstract

Platelet-derived growth factor-BB (PDGF-BB) triggered signal transduction was investigated in human foreskin fibroblasts with endogenous PDGF β-receptors, and porcine aortic endothelial (PAE) cells with stably transfected PDGF β-receptors. Immunoprecipitation and immunoblotting showed that PDGF induced dose-dependent autophosphorylation of PDGF β-receptor, and that PLC-γ associates with autophosphorylated PDGF β-receptors and becomes phosphorylated. Activation of PLC-γ is known to induce fluctuations of the concentration of cytoplasmic calcium ([Ca²⁺]_i). Microfluorometry and digital imaging were employed for measurements of the concentration of [Ca²⁺]_i. In both cell types the growth factor induced four types of [Ca²⁺]_i responses; no rise, a small and sluggish monophasic rise, a biphasic rise with an initial transient peak followed by a sustained elevation, and finally regular oscillations. The frequencies and amplitudes of the oscillatory responses were independent of agonist concentration after stimulation with PDGF-BB. Latency, the period from application of stimulus to the first [Ca²⁺]_i peak, was reduced at higher concentrations of agonist. Also, the proportion of responding cells increased with higher concentrations of ligand. Oscillations of [Ca²⁺]_i were elicited at submaximal concentrations of agonist. In PAE cells PDGF-BB triggered a single [Ca²⁺]_i peak in absence of external Ca²⁺. Ligand-induced oscillations and sustained increases of [Ca²*]_i were counteracted by the inorganic Ca²⁺ channel blocker Ce^{3 +}. These results show that similar types of [Ca²⁺]_i responses occur in different cell types independently of whether the PDGF β-receptors are expressed endogeneously or after transfection. Potentially, the different [Ca²⁺]_i responses have distinct physiological consequences.

Key Words:

• fibroblasts
• endothelial cells
• transfection
• platelet-derived growth factor
• calcium
• phospholipase C-γ