Piper sarmentosum Roxb. Inhibits Angiotensin-Converting Enzyme Activity in Phorbol 12-Myristate-13-Acetate-Induced Endothelial Cells.

Angiotensin-converting enzyme (ACE) plays a crucial role in the pathogenesis of hypertension. Piper sarmentosum Roxb., an herb known for its antihypertensive effect, lacks a comprehensive understanding of the mechanism underlying its antihypertensive action. This study aimed to elucidate the antihypertensive mechanism of aqueous extract of P. sarmentosum leaves (AEPS) via its modulation of the ACE pathway in phorbol 12-myristate-13-acetate (PMA)-induced human umbilical vein endothelial cells (HUVECs). HUVECs were divided into five groups: control, treatment with 200 µg/mL AEPS, induction 200 nM PMA, concomitant treatment with 200 nM PMA and 200 µg/mL AEPS, and treatment with 200 nM PMA and 0.06 µM captopril. Subsequently, ACE mRNA expression, protein level and activity, angiotensin II (Ang II) levels, and angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) mRNA expression in HUVECs were determined. AEPS successfully inhibited ACE mRNA expression, protein and activity, and angiotensin II levels in PMA-induced HUVECs. Additionally, AT1R expression was downregulated, whereas AT2R expression was upregulated. In conclusion, AEPS reduces the levels of ACE mRNA, protein and activity, Ang II, and AT1R expression in PMA-induced HUVECs. Thus, AEPS has the potential to be developed as an ACE inhibitor in the future.

Induction and characterization of extracellular traps by gilthead seabream (Sparus aurata L.) head-kidney leucocytes.

The aim of this study was the induction and characterization of extracellular traps (ETs) produced by gilthead seabream (Sparus aurata L.) head-kidney leucocytes. The cells were incubated several times (10, 30, 60, 120, and 180 min) with different concentrations of the stimulants diluted in RPMI-1640 culture medium: RPMI-1640 (control), ß-glucan from Saccharomyces cerevisiae (BG, 0-400 µg mL-1), lipopolysaccharide from Escherichia coli (LPS, 0-10 µg mL-1), calcium ionophore A23187 (CaI, 0-5 µg mL-1), Phorbol 12-myristate 13-acetate (PMA, 0-1000 ng mL-1) and polyinosinic-polycytidylic acid sodium salt (Poly I:C, 0-200 µg mL-1). BG, LPS and CaI exerted only weak stimulatory activity, while PMA and poly I:C exerted a potent one. After stimulation of the leucocytes, ETs structures were quantified and visualised through staining of the chromatin with nucleic acid-specific dyes and immunocytochemical probing of characteristic proteins expected to decorate the structure. ETs structures had DNA and myeloperoxidase. The ETs morphology was studied by light and scanning electron microscopy. These data confirm that seabream leucocytes form ETs with different morphological properties, depending on the used stimulant. These results will be the basis for new studies to analyse the implication of this mechanism in fish immunity. All this new knowledge will have its application in fish farms when we learn to manipulate the innate immune response in order to mitigate microbial infections.

Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP2), although the structural rearrangements occurring on PIP2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence (121)KRWRK(125), which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP2 facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5'-6'-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP2-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP2 mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4-PIP2 interacting site and conditions that deplete PIP2 or restrict access of TRPV4 to PIP2--in the case of PACSIN3--change tail conformation and negatively affect channel activation by hypotonicity and heat.

Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

BACKGROUND: Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. METHODS AND RESULTS: SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCß knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCß inhibitor LY333531. NADH (30-300 µmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCß small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCß expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCß inhibitor LY333531. CONCLUSIONS: NADH-induced inhibition of endothelial SK channel function is mediated via PKCß. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

Analysis of reproducibility and robustness of OrganoPlate® 2-lane 96, a liver microphysiological system for studies of pharmacokinetics and toxicological assessment of drugs.

Establishing the functionality, reproducibility, robustness, and reliability of microphysiological systems is a critical need for adoption of these technologies. A high throughput microphysiological system for liver studies was recently proposed in which induced pluripotent stem cell-derived hepatocytes (iHeps) and non-parenchymal cells (endothelial cells and THP-1 cells differentiated with phorbol 12-myristate 13-acetate into macrophage-like cells) were co-cultured in OrganoPlate® 2-lane 96 devices. The goal of this study was to evaluate this platform using additional cell types and conditions and characterize its utility and reproducibility. Primary human hepatocytes or iHeps, with and without non-parenchymal cells, were cultured for up to 17 days. Image-based cell viability, albumin and urea secretion into culture media, CYP3A4 activity and drug metabolism were assessed. The iHeps co-cultured with non-parenchymal cells demonstrated stable cell viability and function up to 17 days; however, variability was appreciable both within and among studies. The iHeps in monoculture did not form clusters and lost viability and function over time. The primary human hepatocytes in monoculture also exhibited low cell viability and hepatic function. Metabolism of various drugs was most efficient when iHeps were co-cultured with non-parenchymal cells. Overall, we found that the OrganoPlate® 2-lane 96 device, when used with iHeps and non-parenchymal cells, is a functional liver microphysiological model; however, the high-throughput nature of this model is somewhat dampened by the need for replicates to compensate for high variability.

Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote Caenorhabditis elegans egg-laying circuit activity and behavior.

Activated Gαq signals through phospholipase-Cß and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cß and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cß functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cß, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cß mutants. Phospholipase-Cß mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cß mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cß, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.

Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.

Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-ß1 (TGF-ß1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.

Human Gut Microbiota Metabolism of Dietary Sesquiterpene Lactones: Untargeted Metabolomics Study of Lactucopicrin and Lactucin Conversion In Vitro and In Vivo.

SCOPE: Gut microbiota converts dietary phytochemicals into metabolites and modulates their health effects. The microbial metabolism of dietary terpenoids, as the sesquiterpene lactones of leafy vegetables, is unknown. METHODS AND RESULTS: In vitro fermentation of lactucopicrin, lactucin, and romaine lettuce with gut microbiota from independent donors, show their extensive metabolism through untargeted metabolomics of the fecal incubations. Dehydroxylations and double bond hydrogenations are the main catabolic reactions. Isomers of dihydrolactucopicrin, tetrahydrolactucopicrin, and deoxylactucin, are observed after lactucopicrin metabolism. Tetrahydrolactucin and hexahydrolactucin are also found after lactucin metabolism. Lettuce fermentation shows similar metabolic conversions. Phase II conjugates of most of these metabolites are detected in the urine of healthy volunteers after escarole salad intake. Glucuronides, and sulfates, of dihydrolactucopicrin, tetrahydrolactucopicrin, dihydrolactucin, and deoxylactucin, are detected in the urine although with large inter-subject variability. CONCLUSION: This is the first report on the gut microbiota metabolism of sesquiterpene lactones in humans, and one of the first reports to describe that dietary terpenoids of widely consumed leafy vegetables are extensively catabolized by human gut microbiota. A large inter-subject variation in the metabolism of sesquiterpene lactones also reflects differences in gut microbiota composition. It suggests that inter-individual differences in their health effects should be expected.

Phorbol ester receptors: autoradiographic identification in the developing rat.

Autoradiography with 3H-labeled phorbol dibutyrate was used for the light microscopic detection of phorbol ester receptors in rat fetuses. In 15- and 18-day fetuses, as well as in adult rats, receptors were found to be concentrated in the central nervous system. The localization of receptors in the ventral marginal zone of the fetal neural tube, the lens of the eye, and other sites suggests a role for phorbol ester receptors in cellular process extension and cell-cell interaction.

Tumor-promoting phorbol esters inhibit binding of epidermal growth factor to cellular receptors.

Tumor-promoting phorbol esters and related plant macrocyclic diterpenes inhibit the binding of epidermal growth factor to its receptors on HeLa cells. This effect shows marked structural specificity and correlates with other biological effects of these compounds on mouse skin and in cell culture systems. The active compounds inhibited binding of 125I-labeled epidermal growth factor with a 50 per-cent effective dose in the range of 10(-8) to 10(-9) M. Inhibition appears to be due to a decrease in the number of available epidermal growth factor receptors rather than a change in receptor affinity. These results suggest that certain biologic effects of tumor promoters may result from alterations in the function of cell surface receptors involved in growth regulation.

Receptor-mediated regulation of superoxide production in human neutrophils stimulated by phorbol myristate acetate.

Human neutrophils contain receptors for phorbol myristate acetate (PMA), a complex lipid that induces them to generate superoxide (O (2)). Binding of PMA to these receptors displays specificity, reversibility, and high affinity. The receptor's apparent KD was approximately 0.29 nM and multiple copies (approximately 2.1 +/- 0.6 x 10(5)) were present per neutrophil. We found that the timing and magnitude of the neutrophil's respiratory burst were set independently. The onset of O (2) production occurred after a lag that was inversely proportional to the initial concentration of added PMA. The extent (rate) of O (2) production was directly proportional to the fractional occupancy of the receptor by PMA. Dual regulatory controls, such as those we noted when neutrophils were stimulated by PMA, could afford metabolic stability in the face of transient or low intensity stimuli without compromising quick and powerful responses to larger disturbances.

Inhibition of phorbol ester-receptor binding by a factor from human serum.

The inhibition of receptor binding of [3H]phorbol-12,13-dibutyrate (PDBu) by a factor from human serum was characterized. The serum factor inhibited [3H]PDBu binding in intact monolayer cultures of the rat embryo cell line CREF N and in a subcellular system containing membranes from these cells. Inhibition occurred at both 37 and 4 degrees C and was rapid and reversible. An analysis of [3H]PDBu binding in the presence of the serum factor indicated that inhibition of [3H]PDBu binding by the serum factor was noncompetitive. Using gel filtration to separate the serum factor from free [3H]PDBu, we obtained evidence that the serum factor does not act by binding or trapping the [3H]PDBu. Unlike the phorbol ester tumor promoters, the serum factor alone did not stimulate the release of choline or arachidonic acid from cellular phospholipids, nor did it inhibit the binding of 125I-labeled epidermal growth factor to cellular receptors. The factor did, however, antagonize the inhibition of epidermal growth factor binding induced by PDBu. Sera from pregnant women were, in general, more inhibitory of [3H]PDBu binding than were those from nonpregnant women, which were more inhibitory than those from men. During these studies we found that CREF N cells responded to being grown in the presence of PDBu by partial down regulation of the phorboid receptor. The 50% effective dose for down regulation was 8 nM PDBu, and the maximum effect occurred after 6 h. Taken together, our results indicate that the serum factor inhibits [3H]PDBu binding by a direct physical effect at the level of the phorboid receptors or their associated membranes. It would appear that if this factor acts in vivo, then it might antagonize certain effects of this class of tumor promoters.

Binding of phorbol dibutyrate and epidermal growth factor to cultured human epidermal cells.

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO#1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 X 10(6) sites/cell) and a low-affinity class (affinity constant = 4,880 nM; 7 X 10(7) sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [3H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [3H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.

Presence of specific binding sites for phorbol ester tumor promoters in human epidermal and dermal cells in culture but lack of down regulation in epidermal cells.

The presence of specific binding sites for phorbol esters was demonstrated in human epidermal and dermal cells in culture by assay of binding of [3H]phorbol-12,13-dibutyrate (PDBU) to intact cells. The specificity of the binding was shown by displacement of the binding with biologically active tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate, teleocidin B, and mezerein, but not with inactive derivatives. The equilibrium binding data were analyzed by the Scatchard method and fitted by a straight line to the model of a single class of binding sites. Human epidermal cells bound PDBU with a Kd of 28 nm at 3.7 X 10(6) molecules per cell, while human dermal cells bound PDBU with a Kd of 27 nm at 2.1 X 10(6) molecules per cell. These values were compared with those of epidermal and dermal cells of mice. Although mouse cells showed the same affinity as did human cells, mouse epidermal cells bound one-third as much as human epidermal cells, and mouse dermal cells bound one-fifth as much as human dermal cells. When precultured with unlabeled PDBU for 24 hr, [3H]PDBU binding decreased time dependently in all cells except human epidermal cells. Thus, the binding of phorbol esters to human epidermal cells is unique in that there are a large number of binding sites compared with mouse epidermal cells, and there is no down regulation.

Specific binding of [20-3H]12-deoxyphorbol 13-isobutyrate to phorbol ester receptor subclasses in mouse skin particulate preparations.

[20-3H]12-Deoxyphorbol 13-isobutyrate ([3H]DPB), an inflammatory but relatively nonpromoting analogue of the phorbol ester tumor promoters, bound to mouse skin particulate preparations in a specific, saturable, and reversible manner. Analysis of the binding yielded curvilinear Scatchard plots, consistent with two binding sites present at 0.14 (Site 1) and 1.6 (Site 2) pmol/mg protein and possessing binding affinities of 6.9 and 86 nM, respectively. Structure-activity analysis yielded good correlation (r = 0.94) for a series of 15 diterpene derivatives, including mezerein, between binding affinities at Site 2 and literature values for mouse ear inflammatory potencies. Comparison of binding by [3H]DPB with that by the typical phorbol ester [20-3H]phorbol 12,13-dibutyrate ([3H]PDBU) indicated that PDBU also bound to the sites recognized by [3H]DPB, with affinities of 0.7 and 10 nM, respectively. In addition, a third PDBU binding site was present in mouse skin at 1.9 pmol/mg protein (Site 3) and possessed an affinity of 53 nM. The affinity of DPB for Site 3, determined from competition of [3H]PDBU binding, was 5400 nM. Despite problems in quantitation, the structure-activity relations for Site 3 appeared to differ from those at Site 2 and resembled more closely those expected for complete promoters. Whether the different binding sites represent distinct protein receptors or the same receptor differentially modified remains to be determined.

Metabolism of tritium-labeled 12-O-tetradecanoylphorbol-13-acetate by cells in culture.

The metabolism of [20-3H]-12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) was studied in human and hamster cell cultures. Within 2 to 3 days after its addition to growing or confluent cultures of hamster embryo fibroblasts, no unchanged [3H]TPA remained in the medium as determined by thin-layer chromatography of the chloroform phase obtained by extraction of the medium with chloroform:methanol:H2O. In contrast, little or no metabolism of [3H]TPA occurred under identical conditions in cultures of human fibroblasts. The major metabolite formed from [3H]TPA in hamster cell cultures was [3H]phorbol-13-acetate. with both hamster and human cells, virtually all cell-associated radioactivity was unchanged [3H]TPA.

Characterization of specific binding of [3H]phorbol 12,13-dibutyrate and [3H]phorbol 12-myristate 13-acetate to mouse brain.

[20-3H]Phorbol 12,13-dibutyrate ([3H]PDBU) and [20-3H]-phorbol 12-myristate 13-acetate ([3H]PMA) bound specifically and with high affinity to one class of saturable binding sites in particulate preparations from mouse brain. The dissociation constants for binding of [3H]PDBU and [3H]PMA were 7 nM and 66 pm, respectively. At half-maximal specific binding, binding of [3H]PDBU was 96% specific and binding of [3H]PMA was 91% specific. At saturation, 28 pmol of [3H]PDBU were bound per mg protein. Nonradioactive phorbol 12-myristate 13-acetate blocked > 95% of the specific [3H]PDBU binding. Conversely, nonradioactive phorbol 12,13-dibutyrate blocked > 95% of the specific [3H]PMA binding. The binding sites in mouse brain for [3H]PDBU and [3H]PMA thus appear to be equivalent. In addition to phorbol 12-myritate 13-acetate, seven other biologically active phorbol-related diterpenes inhibited [3H]PDBU binding with potencies which correlated with their biological activities. In the case of phorbol 12,13-diacetate, inhibition of [3H]PDBU binding was shown to be competitive. Phorbol (20 microgram/ml) and 4 alpha-phorbol 12,13-didecanoate (20 microgram/ml), two compounds devoid of biological activity, had no effect on binding. Binding was sensitive to heat, papain, and phospholipase A2 but was insensitive to neuraminidase. Based on the high level of specific phorbol ester binding in brain, we postulate that the phorbol ester target plays a functional rather than information-transducing role in the cell.

Differences in the metabolism of 12-O-[3H]tetradecanoylphorbol-13-acetate and [3H]phorbol-12,13-didecanoate by cells in culture.

The metabolism of the tumor promoters 120-O-[3H]tetradecanoylphorbol-13-acetate ([3H]TPA) and [3H]phorbol-12,13-didecanoate ([3H]PDD) was analyzed in several cell types in culture. In contrast to the rapid metabolism of [3H]TPA, [3H]PDD was degraded much more slowly in hamster, rat, chick, and mouse fibroblasts. Human fibroblasts did not significantly metabolize either phorbol diester over a three-day period. In hamster fibroblasts, addition of increasing amounts of nonradioactive TPA inhibited the metabolism of [3H]TPA, while a 100-fold excess of PDD had no effect on [3H]TPA metabolism. Primary cultures of hamster epidermal cells, a long-term epidermal cell line, and a hamster preadipose cell line rapidly metabolized [3H]TPA but only slowly metabolized [3H]PDD. In contrast to human fibroblasts, a human hepatoma cell line metabolized [3H]TPA, but these cells metabolized [3H]PDD much more slowly. The profile of metabolites produced from [3H]PDD was studied in two cell types. In hamster cells, the major metabolite produced was [3H]phorbol-12-decanoate while in BALB/c 3T3 cells, approximately equal amounts of [3H]phorbol-12-decanoate and [3H]phorbol-13-decanoate were produced. When tested for biological activity in cell culture, phorbol-13-decanoate was 17 to 40 times less active than PDD as measured by the induction of ornithine decarboxylase in hamster cells and the stimulation of 2-deoxyglucose uptake in BALB/c 3T3 cells. Phorbol-12-decanoate was virtually inactive in both assays.

Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain.

The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23 degrees of 3.75 X 10(7) M-1 min-1 and a first-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd for [3H]PDBU binding varied only slightly with temperature. From its temperature dependence, [3H]PDBU binding appeared to be associated with a small increase in enthalpy (delta H degrees = +0.4 kcal/mol) and a large increase in entropy (delta S degrees = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30 degrees to 62 min at 4 degrees. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethyleneglycolbis(beta-aminoethyl ether)N,N'-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.

Specific binding of [3H]phorbol dibutyrate to phorbol diester-responsive and -resistant clones of a human myeloid leukemia (KG-1) line 1.

Phorbol diesters induce macrophage-like differentiation in KG-1 and HL-60 human acute myelogenous leukemia cell lines. We developed a cloned subline of KG-1, known as KG-1a, that does not differentiate when exposed to phorbol diesters. Both KG-1 and KG-1a cells have a single class of specific high-affinity receptors for labeled phorbol-12,13-dibutyrate with a mean Kd of 1.47 +/- 0.10 (S.E.) X 10(-8) M and 0.85 +/- 0.20 X 10(-8) M for the sensitive parental KG-1 line and the resistant KG-1a subline, respectively (p less than 0.025). The number of [3H]phorbol-12,13-dibutyrate binding sites (mean +/- S.E.) per cell was 3.85 +/- 0.98 X 10(5) and 3.94 +/- 0.31 X 10(5) on KG-1 and resistant KG-1a cells, respectively. We observed no significant decrease of specific binding with time (down regulation) in either KG-1, KG-1a, or HL-60 cells, suggesting that down regulation of specific phorbol-12,13-dibutyrate binding is not critical to induction of differentiation. Our data also confirm that the presence of specific high-affinity phorbol receptors on leukemic cells does not assure that phorbol diesters can trigger their differentiation.