Pb 2+ Title

H.A.Godwin, Curr.Opinion Chem.Bio. 5, (2001) M.Mazzolini,S.Traverso,C.Marchetti, J.Neurochem.2001 Oct;79(2): Ca 2+ Pb 2+ Ca 2+ Calcium, a second messenger Pb 2+ Lead, interferes with calcium signalling Uptake machinery Lead action Pb 2+ Ca 2+ ? 2 Lead introduction

Rat cerebellar granule cells Membrane permeant high affinity fluorescent dye: Indo-1 Two-photon laser scanning microscopy (TPLSM) TPEN (100M) Pb 2+ 45´ Indo-1 AM - 1 M 30´ Standard Probe, model and microscopy choice 3

Pb 2+ M.E.Legare, R.Barhoumi, E.Hebert, G.R.Bratton, R.C.Burghardt and E.Tiffany-Castiglioni, Analysis of Pb2+ Entry into Cultured Astroglia, Toxicological Sciences 46, (1998) Figura 5 a) rappresentazione delleccitazione a doppio fotone di una molecola di Indo-1. b) spettro dellIndo-1 a varie concentrazioni di calcio, si nota una lunghezza donda non sensibile alla variazione di tale ione; c) sezioni durto dazione dellIndo-1. In azzurro la sonda libera, in violetto quella legata; d) Spettri relativi della sonda libera (in azzurro), legata al calcio (in violetto) e legata al piombo (in nero). Si evidenziano i picchi a 405nm (punto a) e 485nm (punto b) corrispondenti rispettivamente alla sonda legata al calcio e libera. Inoltre a 460nm (punto c) il punto isosbestico, insensibile alla concentrazione del calcio, ma dipendente da quella del piombo (d). Autofluorescence <<1% Residual Indo1 fluorescence: <5% Indo spectra and binding 4

Indo-1 UV excitation Reduced photoxicity and photodamages High resolution and confocality Wavelength: 700nm ( nm) Mean power on the sample: 5-7mW Pulsed laser: 80MHz ; fs Resolution Axial: 700nm Lateral: 350nm Temporal: 3s/pxl A.Diaspro, Confocal and Two-Photon Microscopy, Wiley-Liss (New York, 2002) F.M. Wahl, Digital Image Signal Processing, Artech House (1987) TPLSM 5

A.Diaspro et al. (1999b), Adapting a compact confocal […], Microsc. Res. Tech. 47, A.Diaspro […] M.Robello and F.Olivini (2001), Two-photon microscopy […], J.Biomed.Opt. Millennia Tsunami (Spectra Physics) PMT1 Microscope TE300Eclipse (Nikon) Scanning Head (PCM2000) PMT2 Microscope 6

Fluorescence intensity time Pb 2+ (20M) Protocol TPEN (100M) KCl 75mM 2 La 3+ (25M) 7

time Fluorescence variations (3.40.5)10 -4 % s -1 mW -2 frm -1 Bleaching rate ~3-8°/ 00 frm -1 T1T1 FLUORESCENCE S0S0 S1S1 PHOSPHO_ RECENCE PHOSPHO_ RECENCE photochemistry quenching photobleaching 2 -0% -10% -20% 08 Time (min) 2 control frames Photobleaching curve Bleaching estimation 64 Photobleaching 8

Brust-Mascher and Webb, Calcium Induced by Large Voltage Pulses in Fish Keratocytes,Biophy.J. Vol m 0255 Intensity (a.u.) Autofluorescence <<1% Mean Indo-1: ~90 m M 50/100 m M Indo1 concentration calibration 9

-75% -60% -45% -30% -15% 0% Time (min) Fluorescence variations (%) Fluorescence variations Photobleaching Pb 2+ (20µM) Time (min) Estimated concentration (µM) Pb 2+ (20µM) ~ bound Lead uptake Var: 54±6% Raw: 67±9% Sat: 63±7% 10

Lanthanum inhibition Pb 2+ La Time (min) Concentration ( M) +La 3+ (25µM) Pb 2+ (20µM) 11

Free cytosolic lead Pb 2+ (20µM) 12

Pb 2+ Rapid lead uptake in the micromolar range (bound) Pb 2+ R.H.S.Westerink et al., Ca2+-indipendent vesicular catecholamine release in PC12 cells by nanomolar concentrations of Pb2+, Journal of Neurochemistry, 2002 Vol Partial lanthanum inhibition (~70%) La 3+ Probable saturation of transport Pb 2+ Free cytosolic lead in the picomolar range C2 (PKC 2pM, sinaptogamin) EF-hand helic (calmodulin, 100pM) Cys 3 (zinc proteins in pM range) Conclusions 13

Aknoledgment

Simulation with MP spectra calcium concentration estimated signal (a.u.) Ratio (405/485) Correlation 460 vs 485 Fluorescence intensity (a.u.) Simulation with theoretical spectra calcium concentration estimated signal (a.u.) Ratio (405/485) freeboundAssym. Dinamic. Estimated Spectra (20nm blue shift + q.y.) obsq.y.shiftest.M.P. low correlation dynamicity HQ405 Wavelength (nm) Fluorescence intensity (a.u.) Filter efficency (%) Bound (est.) Free (est.) Free (MP) Bound (MP) Extras: Indo1 Two-Photon Spectra A1

Extras: Indo1 lead quenching <5% A2

Extras: mathematical model Linear photobleaching time-window Linear quenching regime Negligible autofluorescence A3