Adenylyl cyclase ( IC50 = 41 nM ); Adenylyl cyclase ( EC50 = 0.5 μM )

The Forskolin (Fsk)-treated Mrp4-/- mice shows an increased number of Ki67-positive and cleaved caspase 3-positive ECs, a significant decrease in the amount of pericyte coverage, and a reduced number of empty sleeves. In pups exposed to hyperoxia (75% oxygen) from P7 to P12, the Mrp4-/- mice shows a significant increase in the unvascularized retinal area[3]. The average blood glucose in the healthy rat group is 102.12±1.94 mg/dL, 101.25±3.56 for control group and 103±2.08 in forskolin group. The data shows that glucose levels at the end of the study are lower in forskolin group, with a significant difference according to the statistical tests applied (p=0.03)[6].

In Vitro Kinase Assays[5]
For Jak3 kinase assays, Fsk-treated MT-2 cells were lysed, clarified, and immunoprecipitated using Jak3 antibody as described above. Kinase reactions were carried out as described previously at 30 °C for 20 min. For PKA kinase assays, untreated MT-2 cells were lysed, and Jak3 was immunoprecipitated and bound to PAS beads as described previously. Immunoprecipitated Jak3 was washed with kinase buffer (50 mm Hepes-NaOH (pH 7.4), 10 mm MgCl2, 0.5 mm EGTA, 0.5 mm DTT, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A) and incubated with 200 μm ATP and purified protein kinase A catalytic subunit (PKAc) as indicated in the figure legends. Kinase reactions were carried out at 32 °C for 30 min followed by vigorous washing of the beads with cold kinase wash buffer as described previously. For [γ-32P]ATP radiolabeled kinase assays using recombinant Jak3, Hek293 cells were transfected with wild type (WT) Jak3 or kinase-dead Jak3 K855A using Lipofectamine 2000 according to the manufacturer's instructions. Cells were lysed and immunoprecipitated with Jak3 antibody (described above). Jak3-bound PAS beads were washed three times in cold lysis buffer (described above) followed by kinase buffer. Kinase reactions were initiated by adding 10 μCi [γ-32P]ATP, 10 μm unlabeled ATP, and 1 μg of purified PKAc to Jak3-bound PAS bead reaction mixtures. Kinase reactions were performed at 32 °C for 30 min. Jak3-bound PAS beads were washed three times in radioimmunoassay buffer (10 mm Tris-HCl, pH 7.4, 75 mm NaCl, 20 mm EDTA, 10 mm EGTA, 20 mm Na4P2O7, 50 mm NaF, 20 mm 2-glycerolphosphate, 1 mm p-nitrophenylphosphate, 0.1% Triton X-100) and one time in kinase wash buffer (described above). The reactions were stopped by adding 2× SDS-PAGE sample buffer followed by SDS-PAGE. Coomassie stainable Jak3 bands were excised from the PVDF membrane and subjected to phosphoamino acid analysis.

In 96-well plates, 5×10⁴ cells of either MT-2 or Quiescent Kit 225 are seeded into each well. Afterwards, cells are pretreated for one hour at concentrations of 1, 5, 10, 25, 50, and 100 μM of forskolin or 1% DMSO (vehicle). Following 20 hours of culture at 37°C and IL-2 stimulation, the cells are harvested. A 20-hour treatment of 1% DMSO is given to control cells. [³H]thymidine is pulsed into the cells at a concentration of 0.5 μCi/200 μL during the last 4 hours of incubation. Using liquid scintillation counting, cells are collected onto fiberglass filters for analysis.

Mice: Mice C57BL/6J are employed. established and frequently backcrossed Mrp4-knockout miceto the C57BL/6J mice At postnatal days 4 (P4) and 5, neonatal mice receive an intraperitoneal injection of forskolin (P5). The controls are mice that have been injected with DMSO. After the P6 euthanasia of the treated mice, their retinas are separated for whole-mount immunohistochemistry (IHC). To compare the retinal vascular phenotypes of WT and Mrp4-deficient mice, the optimal concentration of Forskolin was found to be 1.0 μg/50 μL (0.3 mg/kg) at P4 and 1.5 μg/50 μL (0.5 mg/kg) at P5. This was achieved by testing the effects of different Forskolin concentrations on the survival rate and retinal vasculature.

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Rats: Four groups of male Wistar rats, ages 10–14 weeks, with mean weights of 300 g±50 g, are created: eight are kept in good health, and 19 are experimentally made to develop diabetes. For eight weeks, either 6 mg/kg of forskolin per day is given orally to both diabetic and healthy rats as a control. Before and after Forskolin treatment, each group's blood glucose levels are measured. After eight weeks of the prescribed treatment and two weeks following the confirmation of diabetes (three weeks following the induction), the diabetic rats are tested.

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Experimental approach: Male Sprague-Dawley rats were treated with either CCl4 and/or forskolin for 6 consecutive weeks. Serum hepatotoxicity markers were determined, and histopathological evaluation was performed. Hepatic fibrosis was assessed by measuring α-SMA expression and collagen deposition by Masson's trichrome staining and hydroxyproline content. The effects of forskolin on oxidative stress markers (GSH, GPx, lipid peroxides), inflammatory markers (NF-κB, TNF-α, COX-2, IL-1β), TGF-β1 and Hh signalling markers (Ptch-1, Smo, Gli-2) were also assessed. Key results: Hepatic fibrosis induced by CCl4 was significantly reduced by forskolin, as indicated by decreased α-SMA expression and collagen deposition. Forskolin co-treatment significantly attenuated oxidative stress and inflammation, reduced TGF-β1 levels and down-regulated mRNA expression of Ptch-1, Smo and Gli-2 through cAMP-dependent PKA activation. [5]

rat LD50 oral 2550 mg/kg Medicinal Research Reviews., 3(201), 1983 [PMID:6345959]
rat LD50 intraperitoneal 92 mg/kg Journal of Medicinal Chemistry., 31(1872), 1988
mouse LD50 oral 3100 mg/kg Medicinal Research Reviews., 3(201), 1983 [PMID:6345959]
mouse LD50 intraperitoneal 68 mg/kg BEHAVIORAL: CHANGES IN MOTOR ACTIVITY (SPECIFIC ASSAY) Journal of Ethnopharmacology., 3(1), 1981 [PMID:7193263]

[1]. Cell Mol Neurobiol. 2003 Jun;23(3):305-14.

[2]. J Postgrad Med. 2012 Jul-Sep;58(3):199-202.

[3]. Nucleic Acids Res. 2022 Apr 8;50(6):3323-3347.

[4]. Br J Pharmacol. 2016 Nov;173(22):3248-3260.

[5]. J Biol Chem. 2013 Mar 8;288(10):7137-46.

[6]. Br J Pharmacol. 2016 Nov;173(22):3248-3260.

Forskolin is a labdane diterpenoid isolated from the Indian Coleus plant. It has a role as a plant metabolite, an anti-HIV agent, a protein kinase A agonist, an adenylate cyclase agonist, an antihypertensive agent and a platelet aggregation inhibitor. It is a labdane diterpenoid, an acetate ester, an organic heterotricyclic compound, a triol, a cyclic ketone and a tertiary alpha-hydroxy ketone.
Potent activator of the adenylate cyclase system and the biosynthesis of cyclic AMP. From the plant Coleus forskohlii. Has antihypertensive, positive inotropic, platelet aggregation inhibitory, and smooth muscle relaxant activities; also lowers intraocular pressure and promotes release of hormones from the pituitary gland.
Forskolin has been reported in Plectranthus, Apis cerana, and Plectranthus barbatus with data available.
Potent activator of the adenylate cyclase system and the biosynthesis of cyclic AMP. From the plant Coleus forskohlii. Has antihypertensive, positive ionotropic, platelet aggregation inhibitory, and smooth muscle relaxant activities; also lowers intraocular pressure and promotes release of hormones from the pituitary gland.
Potent activator of the adenylate cyclase system and the biosynthesis of cyclic AMP. From the plant Coleus forskohlii. Has antihypertensive, positive inotropic, platelet aggregation inhibitory, and smooth muscle relaxant activities; also lowers intraocular pressure and promotes release of hormones from the pituitary gland.

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1. As initially shown by Seamon and Daly, the diterpene forskolin directly activates adenylyl cyclase (AC) and raises cyclic AMP levels in a wide variety of cell types. In this review, we discuss several aspects of forskolin action that are often unappreciated. These include the utility of labeled forskolin as a means to quantitate the number of AC molecules; results of those types of studies, coupled with efforts to increase AC expression, document that such expression stoichiometrically limits cyclic AMP formation by hormones and neurotransmitters. 2. Response to forskolin is also strongly influenced by the activation of AC by the heterotrimeric G-protein, Gs. Gs-promoted enhancement of AC activity in response to forskolin occurs not only when cells are incubated with exogenously administered agonists that activate G-protein-coupled receptors but also by agonists that can be endogenously released by cells. 3. Such agonists, which include ATP and prostaglandins, serve as autocrine/paracrine regulators of cellular levels of cyclic AMP under "basal" conditions and also in response to forskolin and to agonists that promote release of such regulators. 4. The ability of forskolin to prominently activate cyclic AMP generation has proved valuable for understanding stoichiometry of the multiple components involved in "basal" cyclic AMP formation, in enzymologic studies of AC as well as in defining responses to cyclic AMP in cells within and outside the nervous system.[1]

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Background and purpose: Liver fibrosis is one of the leading causes of morbidity and mortality worldwide with very limited therapeutic options. Given the pivotal role of activated hepatic stellate cells in liver fibrosis, attention has been directed towards the signalling pathways underlying their activation and fibrogenic functions. Recently, the hedgehog (Hh) signalling pathway has been identified as a potentially important therapeutic target in liver fibrosis. The present study was designed to explore the antifibrotic effects of the potent Hh signalling inhibitor, forskolin, and the possible molecular mechanisms underlying these effects. Conclusion and implications: In our model, forskolin exerted promising antifibrotic effects which could be partly attributed to its antioxidant and anti-inflammatory effects, as well as to its inhibition of Hh signalling, mediated by cAMP-dependent activation of PKA.[4]

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Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γc chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.[5]

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Forskolin is a diterpene derived from the plant Coleus forskohlii. Forskolin activates adenylate cyclase, which increases intracellular cAMP levels. The antioxidant and antiinflammatory action of forskolin is due to inhibition of macrophage activation with a subsequent reduction in thromboxane B2 and superoxide levels. These characteristics have made forskolin an effective medication for heart disease, hypertension, diabetes, and asthma. Here, we evaluated the effects of chronic forskolin administration on blood glucose and oxidative stress in 19 male Wistar rats with streptozotocin-induced diabetes compared to 8 healthy male Wistar rats. Rats were treated with forskolin, delivered daily for 8 weeks. Glucose was assessed by measuring fasting blood glucose in diabetic rats and with an oral glucose tolerance test (OGTT) in healthy rats. Oxidative stress was assessed by measuring 8-hydroxydeoxyguanosine (8‑OHdG) in 24-h urine samples. In diabetic rats, without forskolin, fasting blood glucose was significantly higher at the end than at the beginning of the experiment (8 weeks). In both healthy and diabetic rats, forskolin treatment lowered the fasting glucose at the end of the experiment but no effect was found on oral glucose tolerance. The 8-OHdG levels tended to be less elevated in forskolin-treated than in untreated group. Our results showed that chronic administration of forskolin decreased fasting blood glucose levels; however, the reductions of 8-OHdG were not statistically significant.[6]

C22H34O7

410.5

410.23

C, 64.37; H, 8.35; O, 27.28

66575-29-9

47936

White to off-white solid powder

1.2±0.1 g/cm3

519.9±50.0 °C at 760 mmHg

282-232ºC

171.8±23.6 °C

0.0±3.1 mmHg at 25°C

1.552

3.4

3

7

3

29

747

8

O1[C@](C([H])=C([H])[H])(C([H])([H])[H])C([H])([H])C([C@]2([C@@]1(C([H])([H])[H])[C@]([H])([C@]([H])([C@@]1([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])([C@@]12C([H])([H])[H])O[H])O[H])OC(C([H])([H])[H])=O)O[H])=O

OHCQJHSOBUTRHG-KGGHGJDLSA-N

InChI=1S/C22H34O7/c1-8-19(5)11-14(25)22(27)20(6)13(24)9-10-18(3,4)16(20)15(26)17(28-12(2)23)21(22,7)29-19/h8,13,15-17,24,26-27H,1,9-11H2,2-7H3/t13-,15-,16-,17-,19-,20-,21+,22-/m0/s1

[(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-3-ethenyl-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-5-yl] acetate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)