Multisite Interactions Between Pb²⁺ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract

We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb2+‐induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high‐affinity (KA(Pb) = 2.4 × 10−12M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar‐(Ki = 7.1 × 10−9M) and micromolar‐ (K′i = 2.8 × 10−7M) affinity sites, respectively. Activation of PKC by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Ca2+‐deficient, Pb2+‐containing medium, enhances the Pb2+‐induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half‐maximal activation of the secretory response from 7.5 × 10−10 to 5.7 × 10−11M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb2+‐induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb2+‐triggered secretion.

Protein kinase C activity and the relations between blood lead and neurobehavioral function in lead workers.

“…Spectrin is found in mouse brain membranes and undergoes phosphorylation (40), and band 4.9 is also found in the nervous system and in rod photoreceptor cells (41). Extensive experimental evidence also documents the interactions between lead and calcium, especially as relevant to PKC and regulation of nervous system functions (15,18,(42)(43)(44). It would be expected that mechanisms that influence PKC in the brain and erythrocytes are similar, and thus protein phosphorylation in erythrocytes could be a surrogate measure of PKC activity in the brain or, more specifically, of phosphorylation of similar proteins in the central nervous system.…”

“…For example, picomolar concentrations of lead can substitute for micromolar concentrations of calcium in the activation of PKC, as assessed by PKC enzyme activity assays (15). To date, PKC is the only cellular target known to be affected by lead in the range of free lead concentrations of 2 × 10 -11 -3 × 10 -11 M (16)(17)(18). Translocation of PKC from the cytosol to the membrane has been observed in immature microvessels, rat glioma cells, and bovine endothelial cells after exposure to lead (9).…”

Electrophysiologic and behavioral effects of perinatal and acute exposure of rats to lead and polychlorinated biphenyls.

“…PKC activation is essential to LTP in both CA1 and CA3 (47,48). PKC activity is altered by lead in a complex fashion, both activating the enzyme and inhibiting it by competitive and noncompetitive actions (49). The various PKC isozymes are affected differentially (50).…”

Characterizing Golden Eagle Risk to Lead and Anticoagulant Rodenticide Exposure: A Review

“…Lead inhibits uptake of calcium into isolated renal mitochondria and may enter mitochondria as a substrate for a calcium transporter (Kapoor et al 1985). This would be consistent with evidence that lead can interact with calcium binding proteins and thereby affect calciummediated or regulated events in a variety of tissues (Fullmer et al 1985;Goldstein 1993;Goldstein and Ar 1983;Habermann et al 1983;Platt and Busselberg 1994;Pounds 1984;Richardt et al 1986;Rosen and Pounds 1989;Simons and Pocock 1987;Sun and Suszkiw 1995;Tomsig and Suszkiw 1995;Watts et al 1995). Impairments of oxidative metabolism could conceivably contribute to transport deficits and cellular degeneration; however, the exact role this plays in lead-induced nephrotoxicity has not been elucidated.…”