Forskolin from Coleus forskohlii

Product No. F3917

CAS RN 66575-29-9
Synonyms: Coleonol, Colforsin

Product Description

T-cell activation is normally triggered by the interaction of a cell surface receptor to its specific ligand molecule. This binding event triggers the rapid hydrolysis of inositol phospholipids to diacylglycerol and inositol phosphates by phospholipase C (PLC). Diacylglycerol, an allosteric activator of protein kinase C (PKC) and inositol phosphates, which trigger Ca2+ release and mobilization, result in a cascade of additional cellular responses mediating T-cell activation. One of these cellular responses is the production and secretion of interleukin-2 (IL-2). Phorbol 12-myristate 13-acetate, which has a structure analogous to diacylglycerol, can also activate PKC.

Jurkat cells are a leukemic T-cell line known to produce IL-2. Under normal growth conditions, little to no IL-2 is produced in Jurkat cells. PMA, through its activation of PKC, can activate T-cells and stimulate a low-level of IL-2 production. When Jurkat cells are stimulated by PMA and a co-stimulator, such as phytohemagglutinin (PHA), IL-2 production is strongly enhanced.1 PHA by itself can trigger a low level of T-cell activation and IL-2 production by binding non-specifically to the cell surface receptor complex, although the combination of PMA and PHA results in greatly increased IL-2 production.

In T-lymphocytes, the activation of adenylate cyclase by forskolin is known to inhibit the synthesis of IL-2.2 Forskolin appears to interfere with this process by indirectly interfering with the activation of phospholipase C.3 Forskolin activates adenylate cyclase, the enzyme that converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP). cAMP is an activator of protein kinase A (PKA). PKA, however, has been shown to phosphorylate a serine residue on PLC, which is thought to indirectly cause its inactivation.

Phospholipase C is normally activated by phosphorylation at multiple tyrosine residues. Phosphorylation of the serine residue by PKA seems to interfere with activation of PLC by tyrosine phosphorylation.

Storage

Store at Room Temperature

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

Preparation Instructions

Soluble in DMSO (see suitability assay below). For other applications, forskolin may also be solubilized in ethanol.

Storage/Stability

All stock solutions should be stored in the dark at –20 °C

Suitability Assay

2.5 ml of Jurkat cells (1x106 cells/ml) and 2.5 ml fresh culture media (RPMI-1640 + 10% fetal calf serum containing 10 ml/l penicillin-streptomycin) were added to 25 cm2 culture bottles. The following additions were made in duplicate.

a. Control - no additions

b. 1 µg/ml PHA + 10 ng/ml PMA
Add 10 µl PHA stock solution (0.5 mg/ml PHA in filter-sterilized PBS) + 5 µl PMA stock solution (10 µg/ml PMA in DMSO)

c. 0.2 mM Forskolin + 1 µg/ml PHA + 10 ng/ml PMA
Add 10 µl PHA stock solution + 5 µl PMA stock solution + 10 µl forskolin stock solution (100 mM forskolin in DMSO)

After mixing well, the bottles were incubated at 37 °C for 24 hours. After centrifugation, the clarified broth was then tested for IL-2 production by ELISA assay. IL-2 production in the test cultures containing 0.2 mM forskolin was inhibited ≥ 50% compared to the test cultures containing only PHA and PMA.

Results

In the presence of 1 ìg/ml PHA and 10 ng/ml PMA plus 0.2 mM forskolin, production of IL-2 was inhibited at 50% as compared to control cells containing no forskolin.

Product Profile

Molecular Formula: C22H34O7
Molecular Weight: 410.5
Purity: Minimum 98%

Materials

     

 References

  1. Manger, B., et al., J. Clin. Invest. 77, 1501 (1986).
  2. Minakuchi, R., et al., J. Immunol. 145, 2616 (1990).
  3. Park, D.J., et al., J. Biol. Chem. 267, 1496 (1992).

 

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