Pb2+

Pb2+

H.A.Godwin, Curr.Opinion Chem.Bio. 5, 223-227 (2001) M.Mazzolini,S.Traverso,C.Marchetti, J.Neurochem.2001 Oct;79(2):407-16

Ca2+

Pb2+

Ca2+ Calcium, a second messenger

Pb2+ Lead, interferes with calcium signalling

Uptake machinery

Lead action

Pb2+

Pb2+

Ca2+

??2

Lead introduction

Rat cerebellar granule cells

Membrane permeant high affinity fluorescent dye: Indo-1

Two-photon laser scanning microscopy (TPLSM)

Pb2+

Pb2+

Pb2+45´ Indo-1 AM - 1M30´ Standard solution@37°C

3

Pb2+pMKPb 35

F

FfPb bound

0][ FF

FF

Pbfree KPb

1][

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, 90-100 (1998)

nMKCa 230

43 10107000 Pb

Ca

K

K

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 (680-1050nm)Mean power on the sample: 5-7mW Pulsed laser: 80MHz ; 100-200fs

ResolutionAxial: 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, 196-205 A.Diaspro […] M.Robello and F.Olivini (2001), “Two-photon microscopy […]”, J.Biomed.Opt.

Millennia Tsunami(Spectra Physics)

PM

T1

MicroscopeTE300Eclipse

(Nikon)

Scanning Head (PCM2000)

PM

T2

Microscope

6

Flu

ore

scen

ce in

ten

sity

time

Pb2+ (20M)

Protocol

TPEN (100M)

KCl 75mM

2‘

La3+ (25M)

7

time

Flu

ore

scen

ce v

ari

ati

ons

(3.40.5)10-4 % s-1 mW-2 frm-1

Bleaching rate ~3-8°/00 frm-1

0

0][

tF

BQ

tF

FPb bound T1

FLU

OR

ESCEN

CE

FLU

OR

ESCEN

CE

S0

S1

PHOSP

HO_

REC

ENCE

PHOSP

HO_

REC

ENCE

photochemistry

quenching photobleaching

tqf

f

KKK

K

QKd

1

10

2

-0%

-10%

-20%

0 8

Time (min)

2 control framesPhotobleaching curveBleaching estimation

64

Photobleaching

8

Brust-Mascher and Webb, “Calcium Induced by Large Voltage Pulses in Fish Keratocytes”,Biophy.J. Vol75 1998

5m

0 255Intensity (a.u.)

1][ IndoPb bound

Autofluorescence <<1%Mean Indo-1: ~90M

50/100M

9

-75%

-60%

-45%

-30%

-15%

0%

0 2 4 6 8

Time (min)

Fluore

scence

vari

ati

ons

(%)

Fluorescence variations

Photobleaching

Pb2+(20µM)

5

15

25

35

45

55

65

75

0 2 4Time (min)

Est

imate

d c

once

ntr

ati

on (

µM

)Pb2+ (20µM) Pb2+ (20µM)

~bound

Lead uptake

Var: 54±6%Raw: 67±9%Sat: 63±7%

10

Pb2+

La3+

FFf

Pbbound

0]

[

5

15

25

35

45

55

65

75

0 2 4Time (min)

Con

cent

ratio

n (

M)

+La3+ (25µM)

Pb2+ (20µM) Pb2+ (20µM)

11

FFF

F

Pb

free

KPb

1]

[

Pb2+ (20µM) Pb2+ (20µM)

12

Pb2+

Pb2+

Pb2+

Pb2+

Pb2+

Pb2+

Pb2+

Rapid lead uptake in the micromolar range (bound)

Pb2+

R.H.S.Westerink et al., “Ca2+-indipendent vesicular catecholamine release in PC12 cells by nanomolar concentrations of Pb2+”, Journal of Neurochemistry, 2002 Vol.80 861-873

Partial lanthanum inhibition (~70%)

La3+

Probable saturation of transport

Pb2+

Pb2+

Free cytosolic lead in the picomolar range

C2 (PKC 2pM, sinaptogamin)

EF-hand helic (calmodulin, calcineurin @ 100pM)

Cys3 (zinc proteins in pM range)

Conclusions

13

Aknoledgment