1. Measurement of intracellular Ca2+ concentration
use fluorescent Ca2+ sensitive dyes
eg INDO-1, FURA-2
light is shone at a cell containing one of these dyes

2. Use of fluorescent Ca2+ dyes
Excitation
light
Emitted
light
At some wavelengths the fluorescence increases
with increased [Ca2+]
At other wavelengths the fluorescence decreases
with increased [Ca2+]
The ratio of fluorescence at two appropriate
wavelengths gives the intracellular [Ca2+]

3. IP3 releases Ca2+ +
IP3
Ca2+

4. Intracellular Ca2+ levels in response to receptor activation
normal extracellular Ca2+
0.8
zero extracellular Ca2+ 2+
0.6
conc intracellular Ca
0.4
0.2
0.0 2 4 6 8
time (min)
Bradykinin

5. IP3 mediated calcium release
does not occur continuously but rather in a series of bursts
this is because calcium itself can cause the opening of calcium channels in response to the initial small release
this leads to an oscillatory response

6. Calcium release in response to carbachol
0.5 2+
0.4
0.3
intracellular conc Ca
0.2
0.1 20 40 60 80
100
120
time (seconds)

7. There are other ways by which Ca2+ can be mobilized from internal stores
1. Skeletal muscle
sarcoplasmic
reticulum
voltage
Ca2+
Ca2+
channel
ryanodine
receptor

8. Ca2+ Mobilization II 2. Cardiac muscle sarcoplasmic reticulum voltage
ryanodine
receptor

9. More Notes some Ca2+ pools only have ryanodine receptors
proof ? ryanodine receptors :-
stimulated by caffeine
inhibited by ruthenium red
some Ca2+ pools only have IP3 receptors
some have BOTH
some have NEITHER??
this area is COMPLEX and not fully understood

10. Many transmitters / hormones stimulate IP3 production
examples are :-
a1 adrenoceptors
bradykinin receptors
M1 and M3 muscarinic receptors
Note - whenever IP3 is produced so is diacylglycerol (DAG)