Lead induced rise in intracellular free calcium is mediated through activation of protein kinase C in osteoblastic bone cells

Abstract

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.

NADPH Oxidase-Mediated Redox Signaling: Roles in Cellular Stress Response, Stress Tolerance, and Tissue Repair

“…As with PKC, increased intracellular Ca 2ϩ has been observed in cells exposed to cytotoxic chemicals (Stoll and Spector, 1993;Liu and Huang, 1997;Yeh et al, 2007;Pourrut et al, 2008), osmotic challenges (Cazalé et al, 1998;Yellowley et al, 2002), physical stretches (Davis et al, 1992;Sokabe et al, 1997), hypoxia (Berna et al, 2001), and inflammatory mediators (Chen et al, 1997;García et al, 1999). It is noteworthy that an increase in intracellular Ca 2ϩ can be a secondary response after PKC activation and vice versa (Schanne et al, 1997;Barnett et al, 2007). The Nox5 isoform contains multiple EF hand Ca 2ϩ binding domains in the N-terminal region, allowing its activation through calcium sensing (Bá nfi et al, 2001).…”

“…It was also demonstrated that exposure of mouse cortical brain slices to hypoosmosis stimulated ROS production, which was absent in brain slices from p47phox knockout animals, suggesting that PKC-induced p47phox phosphorylation had a pivotal role in osmotic stress-induced Nox activation in astrocytes (Reinehr et al, 2007). It is noteworthy that several lines of study have demonstrated that PKC activation can be observed after exposing cells to a variety of stress stimuli (for example, cytotoxic chemicals, physical stretch, osmotic shock, hypoxia, and inflammatory cytokines) (Kugiyama et al, 1992;Rzymkiewicz et al, 1996;Goldberg et al, 1997;Liu and Huang, 1997;Schanne et al, 1997;Wyatt et al, 1997;Deng and Poretz, 2002;Motley et al, 2002;Suzuma et al, 2002;Yeon et al, 2002;Liu et al, 2003), and PKC may be viewed as a stress sensor in some circumstances (Barnett et al, 2007). If PKC is indeed involved in Nox activation in acute stress responses, the specific roles of different PKC isoforms need to be defined.…”

Suppression of Proteoglycan Synthesis by Calcium Ionophore A23187 in Cultured Vascular Endothelial Cells: Implication of Intracellular Calcium Accumulation in Lead Inhibition of Endothelial Proteoglycan Synthesis.

“…9,30) Calcium ionophore A23187 also modulates the proliferation of vascular endothelial and smooth muscle cells in a similar manner to that of lead. 30) Lead characteristically perturbs the calcium-dependent intracellular regulation pathway by increasing the intracellular calcium level and/or mimicking calcium in various cell types, [12][13][14][15][16][17] although the metal can be an effective antagonist of calcium transients under certain conditions. [31][32][33] In the present study, it was shown that A23187 suppresses the synthesis of proteoglycans, and lead increases the accumulation of intracellular calcium, suggesting that lead inhibition of endothelial proteoglycan synthesis 7,8) is mediated by the intracellular calcium increased by the metal.…”

“…7) The inhibition causes a lower response of the cells to endogenous bFGF, 8) resulting in an inhibition of proliferation 9) and repair 10) after damage of the cell layers. 11) Since lead can increase intracellular calcium levels in various cell types [12][13][14][15][16][17] and can substitute for calcium in the intracellular signaling, 18,19) we also hypothesized that lead inhibition of endothelial proteoglycan synthesis may partly be due to an increase in intracellular calcium. In the present study, we investigated the effect of calcium ionophore A23187 on proteoglycan synthesis and that of lead on the accumulation of intracellular calcium in cultured vascular endothelial cells.…”

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“…In most of the previous studies, the harmful effects of lead were noted 2,3,4 . Many mechanisms are proposed for lead-induced hypertension, including alteration in calcium (Ca 2+ ) flux, lowering the calcium binding capacity in intracellular Ca 2+ concentration 5,6 , inhibition of sodium pump 7 , increased activity of renin-angiotensin system 8 , altered kallikreinkinin system causing decreased plasma levels of bradikinin 9,10 and increased cardiovascular sensitivity to endogeneous substances such as catecholamines 11 . Lead exposure results in oxidative stress and inflammation which in turn lower bioavailability of NO and promote hypertension, endothelial injury/ dysfunction and cardiovascular disease.…”