PKC/protein kinase C

In OK cells, phorbol 12,13-dibutyrate (Phorbol dibutyrate) (1 mM) inhibits Na/K-ATPase transport activity and activates PKC [3].

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Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the Phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). Researchers determined the effects of PDBu (10(-7) M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. Researchers used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10(-6) M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10(-6) M). Researchers also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined. [1]

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Researchers have measured the dissociation rate of phorbol 12-myristate 13-acetate (PMA), a potent tumor promoter, Phorbol 12,13-dibutyrate (PDBu), a weak tumor promoter, and 12-deoxyphorbol 13-phenylacetate (dPP), an antitumor promoter, from intact mouse keratinocytes. PDBu and dPP showed a very rapid release from the cells (t1/2 = 1 min), whereas PMA showed a slower release (t1/2 = 9 min). Western blot analysis of the amounts of protein kinase C alpha (PKC alpha) and PKC delta in the soluble fraction and the Triton X-100-soluble particulate fraction revealed that translocation of both isozymes from the soluble to the particulate fraction was reversible when the phorbol esters were washed off. Washes of 5-15 min resulted in complete redistribution of the PKC isozymes when the cells were previously treated with 1 microM dPP or 1 microM PDBu for 5 min. In the case of treatment with 100 or 10 nM PMA, the redistribution required a longer time; nevertheless, the PKC isozymes returned to the soluble fraction within 60 min. Longer initial treatments with PMA, dPP, and PDBu (up to 60 min) translocated PKC in a very similar, completely reversible fashion. Researchers conclude that in this cell line phorbol esters do not induce the conversion of PKC isozymes to an integral membrane state. [2]

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Na/K-ATPase in renal epithelium is expressed at the basolateral surface and thus is critical for vectorial solute transport. One potential mode of regulation of Na/K-ATPase involves the intracellular effector protein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester Phorbol 12,13-dibutyrate (PDBu) (1 microM) inhibited Na/K-ATPase transport activity in OK cells (Vmax decreased 42%; p < 0.02), but not in LLC-PK1 cells. By immunoblot, both cell types expressed detectable levels of PKC alpha and PKC sigma. In response to PDBu, PKC alpha translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of 32PO4 in multiple substrates in both cell types, but a approximately 109-kDa substrate with neutral pI was detected only in the OK cell. Anti-LEAVE, directed against a highly conserved sequence in the H4-H5 loop of all known alpha isoforms of Na/K-ATPase, recognized a approximately 109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a protein that comigrated with the large phosphoprotein which was only present in OK cells. Following 32PO4 loading and PDBu treatment, anti-LEAVE immunoprecipitated a approximately 109-kDa phosphoprotein in OK but not LLC-PK1 cells. These data support the notion that PKC is capable of phosphorylating the alpha subunit and inhibiting Na/K-ATPase transport activity in intact renal cells. Furthermore, they suggest that some forms of Na/K-ATPase in the kidney are not susceptible to PKC phosphorylation and that this heterogeneity may contribute to response diversity. [3]

To explore the role of protein kinase C (PKC) in the augmented bronchial smooth muscle (BSM) contraction observed in the antigen-induced airway hyperresponsive (AHR) mice, the effects of a PKC activator, Phorbol 12,13-dibutyrate (PDBu), on BSM contraction were compared between the AHR and control mice. Actively sensitized mice were repeatedly challenged by antigen inhalation. Twenty-four hours after the final antigen challenge the isometrical contractions of the BSMs were measured. The BSM contraction induced by acetylcholine, but not high K(+) depolarization, was significantly augmented in the AHR mice. In BSMs of control mice, PDBu caused a significant increase in tension when the tissues were precontracted with high K(+), although PDBu itself had no effect on basal tone. The PDBu-mediated contraction was markedly augmented in BSMs of the AHR mice. These findings suggest that an increase in the PKC-mediated signaling is involved in the augmented contraction of BSMs in the antigen-induced AHR mice. [4]

The SMCs were then harvested by filtration through the Nitex mesh. The filtrate containing the cells was centrifuged at 350 g for 10 min at room temperature. The cells in the pellet were resuspended in DMEM growth medium with 5% fetal bovine serum, 5% penicillin-streptomycin, 50 μg/ml gentamicin, and 2 μg/ml amphotericin B on Lab-Tek II chamber slides at 37°C and 5% CO2 in an incubator with regulated humidity. The cell-attached medium was then replaced with serum-free medium for 24 h before treatment of the SMCs with Phorbol 12,13-dibutyrate (PDBu) (10−7 M for 20 min) before and after calphostin C 10−6 M, C3 exoenzyme 2.5 μg/ml, or Y 27632 10−6 M for 1 h. For controls, the cells were left untreated with any agent but otherwise under the similar conditions as the treated cells. (Cell passage was carefully recorded and the cells were not used beyond second passage to eliminate the concern of the SMC dedifferentiation in culture medium after a number of passages.) [1]

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Western blot analysis.[1]
Levels of pThr696-MYPT1 and pThr18/Ser19-MLC20 were determined via Western blot analysis of human IAS SMCs, under control and following pretreatment with Phorbol 12,13-dibutyrate (PDBu) (10−7 M), before and after calphostin C (10−6 M), C3 exoenzyme (2.5 μg/ml), and Y 27632 (10−6 M) conditions. The cells were rinsed with PBS and lysed with 500 μl lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, pH 7.4) containing protease and phosphatase inhibitor cocktail was added to inactivate proteases and phosphatases. Cell lysate was centrifuged at 14,000 rpm for 5 min and supernatant was transferred into different tubes and protein quantification was carried out by using BCA kit from Pierce. Twenty micrograms of protein in 20 μl of lysates were mixed with 2× Laemmli sample buffer (with final concentrations of 62.5 mM Tris, 1% SDS, 15% glycerol, 0.005% bromophenol blue, and 2% β-mercaptoethanol) and placed in a boiling water bath for 5 min. Samples were loaded on 15% polyacrylamide gel. The separated proteins were electrophoretically transferred onto a 0.2 μm Immun-Blot polyvinylidene fluoride membrane). The membranes were kept in Odyssey blocking buffe for 1 h and stained overnight with the primary antibody (raised in goat) of pThr696-MYPT1 pThr18/Ser19-MLC20 in Odyssey blocking buffer containing 0.2% Tween. The membranes were washed thrice for 10 min each with PBS with 0.2% Tween and incubated with anti-goat infrared dye (IRdye800)-conjugated secondary antibody for 1 h and the membranes were scanned with Odyssey infrared scanner. Western blot band intensities were calculated with Image J 1.41o (National Institutes of Health; means ± SE), and graphs were plotted as the intensity ratios of pThr696-MYPT1 and pThr18/Ser19-MLC20, vs. nonphosphorylated forms of MYPT1 and MLC20.[1]

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For the assessment of the amounts of soluble and membrane-associated PKCa and PKCG, the cells were treated with the indicated doses of PMA, Phorbol 12,13-dibutyrate (PDBu), and dPP for 5, 15, or 60 min at 37 “C; washed with three changes of the same medium at the same temperature; and finally rinsed twice with ice-cold phosphate-buffered saline (without Ca2+ and MP, pH 7.4). The cells were harvested and lysed, and the soluble and Triton X-100-soluble particulate fractions were prepared as described previously. In the case of PDBu and dPP treatments, the washing buffer and lysis buffer contained the same concentration of PDBu or dPP as was present during the initial incubation because of the very rapid dissociation rate of these ligands (see below). The protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. [2]

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Immunoprecipitation-Confluent 60-mm dishes of OK and LLCPK, cells were loaded with radioactive tracer under the same conditions indicated above for intact cell phosphorylation. Monolayers were treated with either Phorbol 12,13-dibutyrate (PDBu) (1 pM) or Me's0 for 10 min followed by three washes of iced Krebs buffer. After adding 250 pl/dish of precipitating buffer (50 mM Tris-HC1, 1% Nonidet P-40,5 mM EDTA, 0.15 M NaCI, pH 8.3) cells were scraped and homogenized 20 strokes in a glass-on-glass homogenizer. The suspension was incubated on ice for 20 min and centrifuged 30 min at 50,000 X g. Amounts of supernatant protein were equalized based on trichloracetic acidprecipitable counts and averaged -35 X lo6 counts/min/sample for OK cells and -7 X lo6 counts/min/sample for LLC-PK1 cells. Homogenates were cleared with rabbit serum (1:lOO) for 60 min at 4 "C followed by 10% (v/v) washed Pansorbin for 60 min. The cleared supernatant was incubated overnight with affinity-purified anti-LEAVE (1:lOO). A secondary goat anti-rabbit IgG antibody (1:100) was added at 4 "C for 60 min, and immune complexes were again precipitated with 10% washed Pansorbin. The pellet was resuspended, vortexed, and washed four times with wash buffer (50 mM Tris-HC1, 0.15 M NaC1, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.3) at 4 "C. After the final wash, the pellet was suspended in SDS sample buffer, boiled for 5 min, and cleared by low speed centrifugation. The proteins were separated on 9% SDS-PAGE and analyzed with autoradiography.[3]

[1]. Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol. 2011 Aug;301(2):G317-25.

[2]. Dissociation of phorbol esters leads to immediate redistribution to the cytosol of protein kinases C alpha and C delta in mouse keratinocytes. J Biol Chem. 1994 Nov 4;269(44):27159-62.

[3]. Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells. J Biol Chem. 1993 Jul 25;268(21):15958-64.

[4]. Augmented PDBu-mediated contraction of bronchial smooth muscle of mice with antigen-induced airway hyperresponsiveness. J Smooth Muscle Res. 2010;46(5):259-66.

Phorbol-12,13-dibutyrate is a white solid. (NTP, 1992)
Phorbol 12,13-dibutanoate is a phorbol ester, a butyrate ester and a tertiary alpha-hydroxy ketone.
A phorbol ester found in CROTON OIL which, in addition to being a potent skin tumor promoter, is also an effective activator of calcium-activated, phospholipid-dependent protein kinase (protein kinase C). Due to its activation of this enzyme, phorbol 12,13-dibutyrate profoundly affects many different biological systems.

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Immunocytochemical studies provide evidence for the activation of RhoA/ROCK by their translocation to the periphery of the SMCs by Phorbol 12,13-dibutyrate (PDBu). The increased translocation of RhoA caused by PDBu was selectively blocked by RhoA inhibitor C3 exoenzyme. In addition, PDBu-induced ROCK II translocation was selectively reversed by C3 exoenzyme and Y 27632. These observations are consistent with a number of recent studies showing the involvement of RhoA/ROCK activation in PDBu-induced actions in different systems. In addition, earlier studies in the rat IAS have shown that contraction of the SMC via direct activation by PKC is dependent on RhoA/ROCK activation. This was shown via the determination the effect of PKC and RhoA/ROCK inhibitors on the PKC-induced contraction of the SMC. In the human IAS SMCs, however, it appears that RhoA/ROCK activation via PDBu appears to be independent of PKC activation. In the present studies immunocytochemical evidence combined with the functional data suggest that PDBu-induced contraction of the human IAS SMC utilize RhoA/ROCK pathways rather than the PKC.
The exact mechanism of RhoA/ROCK activation following Phorbol 12,13-dibutyrate (PDBu) that is independent on PKC activation is not presently known. It is well known that PDBu can also bind to the proteins other than PKC which has C1 domain (DAG binding domain). These include GTPase-activating proteins for Rac, Ras guanyl-releasing proteins (RasGRPs), and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK), a member of Rho family G proteins. The fate of PDBu-activated PKC that does not participate in RhoA/ROCK stimulation is not presently known. In addition, whether overactivation of PKC by PDBu leads to its own downregulation or disintegration or whether it activates other unknown intermediary kinase/s for RhoA/ROCK activation remains to be determined.
We conclude that Phorbol 12,13-dibutyrate (PDBu)-induced contraction of the human IAS SMC appears to be associated with RhoA/ROCK activation. The relative contribution of PKC vs. RhoA/ROCK pathways in the basal IAS tone in the human IAS remains to be determined.[1]

C28H40O8

504.6124

504.272

C, 66.65; H, 7.99; O, 25.36

37558-16-0

37783

White to light yellow solid powder

1.27g/cm3

623.4ºC at 760mmHg

199.3ºC

1.574

2.632

3

8

9

36

1030

8

CCCC(=O)O[C@@H]1[C@H]([C@]2([C@@H](C=C(C[C@]3([C@H]2C=C(C3=O)C)O)CO)[C@H]4[C@@]1(C4(C)C)OC(=O)CCC)O)C

BQJRUJTZSGYBEZ-YVQNUNKESA-N

InChI=1S/C28H40O8/c1-7-9-20(30)35-24-16(4)27(34)18(22-25(5,6)28(22,24)36-21(31)10-8-2)12-17(14-29)13-26(33)19(27)11-15(3)23(26)32/h11-12,16,18-19,22,24,29,33-34H,7-10,13-14H2,1-6H3/t16-,18+,19-,22-,24-,26-,27-,28-/m1/s1

Phorbol 12,13-dibutanoate

PDBu; Phorbol 12,13-dibutyrate; Phorbol 12,13-dibutyrate; 37558-16-0; PDBU; Phorbol dibutyrate; Phorbol 12,13-dibutanoate; Phorbol-12,13-dibutyrate; PHORBOL12,13-DIBUTYRATE; (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulene-9,9a-diyl dibutanoate; Phorbol dibutyrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 125 mg/mL (~247.72 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.12 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 20.8 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: ≥ 2.08 mg/mL (4.12 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.12 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 20.8 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.)