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Core D, San Francisco: Laboratory for Development of Signaling Assays. B Lymphocytes Initiate ligand screen, 1st publication (with Core C, Dallas) Long term culture Myocytes. The SF VAMC AfCS Lab. Tim O’Connell. Paul Simpson. Luyi Li. Bill Seaman. Tamara Roach. Melissa

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Core d san francisco laboratory for development of signaling assays
Core D, San Francisco: Laboratory for Development of Signaling Assays

  • B Lymphocytes

    • Initiate ligand screen, 1st publication (with Core C, Dallas)

    • Long term culture

  • Myocytes


The SF VAMC Signaling Assays

AfCS Lab

Tim

O’Connell

Paul

Simpson

Luyi

Li

Bill

Seaman

Tamara

Roach

Melissa

Kachura

Susan

Ricker


Myocyte isolation procedure

Myocyte Isolation Procedure Signaling Assays

Hanging Heart

In Situ Perfusion

Steps in both:

• Ca++ wash out

• Collagenase digestion

( 50 mM Ca++)

• Mechanical disaggregation

• Collagenase inhibition (BCS)

• Ca++ reintroduction

• Wash & count

Constant Flow

Constant

Pressure

or

Constant Flow

pump

pump

  • • needle inserted in LV apex in situ

  • • drain atrium & clamp aorta

  • • constant pressure (~75 mmHg, 125 cm)

  • or

  • constant flow (4 ml/mim)

• dissect heart

• cannulate aorta

• constant flow (4 ml/min)


Myocyte yields with different isolation techniques
Myocyte Yields with Different Signaling AssaysIsolation Techniques

* measured since January, 2002

In Situ preparation is much easier technically

In Situ constant flow preparation is easier than constant pressure


Myocyte culture procedure
Myocyte Culture Procedure Signaling Assays

Goal: Maintain rod-shaped myocytes that signal for 72 hrs

Plate for 1 hr on laminin coated dishes in:

MEM w/Hanks BSS w/

5% BCS

10 mM BDM

Penicillin

Change Medium to:

MEM w/Hanks BSS w/

1 mg/ml Insulin

0.5 mg/ml Transferrin

0.55 ng/ml Selenium

1 mg/ml BSA

10 mM BDM

Penicillin

Culture for up to 72 hours at 37°C in 2% CO2

0 hr

24 hr

72 hr


Myocyte experimental timeline
Myocyte Experimental Timeline Signaling Assays

Plating

Assay

Signaling

At 24 hrs

Assay

Signaling

At 72 hrs

Myocyte

Isolation

Medium

Change

3 hrs

1 hr

24 hrs

48 hrs

Assays:

Gs: cAMP, PLB phosphorylation, myocyte contraction

Gi:inhibition of cAMP

Gq:ERK phosphorylation


Activation of gs signaling in myocytes at 24 and 72 hours

20000 Signaling Assays

16000

12000

8000

4000

0

1

0

-

1

0

1

0

-

9

1

0

-

8

1

0

-

7

1

0

-

6

1

0

-

5

Activation of Gs Signaling in Myocytes at 24 and 72 Hours

Isoproterenol, a b-AR agonist that signal through Gs,

increases cAMP in a concentration-dependent manner

24 hrs

EC50 28 nM

fmol cAMP/

20,000 myocytes

72 hrs

EC50 24 nM

Isoproterenol (nM)


Activation of gi signaling in myocytes at 24 and 72 hours

120000 Signaling Assays

100000

80000

35000

30000

25000

20000

15000

10000

5000

0

Activation of Gi Signaling in Myocytes at 24 and 72 Hours

Carbachol, a muscarinic agonist that signals through Gi,

reduces isoproterenol- and forskolin- induced cAMP accumulation

Isoproterenol (1 mM)

Forskolin (100 mM)

Carbachol (100 mM)

fmol cAMP/

20,000 myocytes

24 hrs

72 hrs

Fsk

Iso

Carb

Fsk

Carb

Control

Iso


Activation of gq signaling in myocytes at 24 and 72 hours
Activation of Gq Signaling in Signaling AssaysMyocytes at 24 and 72 Hours

Phenylephrine, an a1-AR agonist, and Endothelin-1 which

both signal through Gq, increase ERK1/2 phosphorylation

24 hrs

72 hrs

Control

PE

20 mM

ET-1

100 nM

PMA

100 nM

Control

PE

20 mM

ET-1

100 nM

PMA

100 nM

Phospho-ERK

Total-ERK


Phospholamban phosphorylation in myocytes at 24 and 72 hours
Phospholamban Phosphorylation in Signaling AssaysMyocytes at 24 and 72 Hours

Isoproterenol, a b-AR agonist that signals through Gs,

increases phospholamban phosphorylation

24 hrs

72 hrs

Control

Iso

1 mM

Control

Iso

1 mM

Gb

Phospho-PLB


Activation of e c coupling in myocytes at 24 hours
Activation of E-C Coupling in Signaling AssaysMyocytes at 24 Hours

Myocytes contracting under field stimulation

Myocytes quiescent for first 5 seconds

Stimulated at 80V, 1 Hz for 20 seconds

Then increase frequency to 1.5 Hz for 15 seconds


Activation of e c coupling in myocytes at 24 hours1
Activation of E-C Coupling in Signaling AssaysMyocytes at 24 Hours

Isoproterenol, a b-AR agonist that signals through Gs

and increases phospholamban phosphorylation,

induces myocyte contraction


Activation of e c coupling in myocytes at 72 hours

8 Signaling Assays

6

4

2

0

Activation of E-C Coupling in Myocytes at 72 Hours

Isoproterenol induces myocyte contraction

Myocyte Contraction measured as %Shortening

of individual cardiac myocytes

*

% Shortening

270%

16

16

Control

1 mM Isoproterenol

*

p < 0.05


Requirements for the ligand screen
Requirements for the Ligand Signaling AssaysScreen

Assay Time Points Myocytes

Phosphoprotein 0, 2, 5, 12, 30 min 5 x 35 mm dish

(250,000 myocytes)

RNA Array 0, 30, 120, 240 min 16 x 60 mm dish

(2,400,000 myocytes)

cAMP 0, 2, 5, 12, 30 min 5 x 35 mm dish

(250,000 myocytes)

Calcium in development

Total 2.9 x106 myocytes

Project that we need 3 hearts/ligand


Summary myocytes
Summary: Myocytes Signaling Assays

  • Criteria

    • Acute signaling: cAMP, phosphorylation, contraction.

    • Suitable for mutation (RNAi, antisense, transfection, etc).

    • Reproducible within and between labs.

    • Convenient, sufficient throughput.

    • Mouse, normal, adult.


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