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Journal of Neurophysiology Oct 19921. The electrophysiological properties of the sensory neurons that mediate withdrawal reflexes in Aplysia are modulated by a number of second messengers. For example,...
1. The electrophysiological properties of the sensory neurons that mediate withdrawal reflexes in Aplysia are modulated by a number of second messengers. For example, the second messengers adenosine 3',5'-cyclic monophosphate (cAMP) and arachidonic acid modulate the S-K+ current (IK,S) and the calcium-activated K+ current (IK,Ca). Recent evidence suggests that protein kinase C (PKC) may also be an important regulator of cellular plasticity. In the present study we examined the possibility that IK,Ca was modulated by the activation of PKC in the pleural sensory neurons. 2. In voltage-clamped sensory neurons the application of phorbol esters, such as phorbol dibutyrate (PDBu), phorbol myristate (PMA), and phorbol diacetate (PDAc), which activate PKC, caused a dose-dependent increase in a voltage-dependent current with properties that resembled IK,Ca. The inactive isomer of phorbol ester, 4 alpha-phorbol, was without effect. 3. This phorbol ester-sensitive current had the kinetics and pharmacological sensitivity of IK,Ca. The current developed slowly during step depolarizations, showed little inactivation, and was activated at membrane potentials greater than approximately 0 mV. In addition, the current modulated by phorbol esters was blocked by a concentration of tetraethylammonium (TEA) that blocks a component of IK,Ca in the sensory neurons. 4. IK,Ca, which was activated directly by the iontophoretic injection of Ca2+, was also enhanced by PDBu. Moreover, the enhancement of Ca(2+)-elicited responses by PDBu persisted after Ca2+ influx was blocked by cobalt. These results indicate that at least one component of the modulation of IK,Ca by PDBu was independent of the modulation of voltage-dependent Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Aplysia; Calcium; Dose-Response Relationship, Drug; Enzyme Activation; Ganglia; In Vitro Techniques; Isoquinolines; Kinetics; Membrane Potentials; Neurons, Afferent; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Piperazines; Potassium Channels; Protein Kinase C; Protein Kinase Inhibitors; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tetraethylammonium; Tetraethylammonium Compounds
PubMed: 1432069
DOI: 10.1152/jn.1992.68.4.1079 -
The American Journal of Physiology Nov 1985In rabbit proximal colon, in vitro addition of phorbol 12,13-dibutyrate (PDB, 10(-7) M) to the serosal bathing medium inhibits mucosal (m)-to-serosal (s) unidirectional...
In rabbit proximal colon, in vitro addition of phorbol 12,13-dibutyrate (PDB, 10(-7) M) to the serosal bathing medium inhibits mucosal (m)-to-serosal (s) unidirectional Na flux (JsmNa) without altering JsmNa or unidirectional Cl fluxes. Similar results were obtained when amiloride (2 X 10(-4) M) was added to the mucosal bathing medium. No additivity of effect was seen when tissues were exposed to both agents. Measurements with carboxyfluorescein reveal that the two agents cause equal decreases of intracellular pH (pHi), an effect that is dependent on the presence of extracellular Na (Na replacement also decreases pHi). No additivity of pHi effects is seen when both agents are added together. To determine the membrane site of this PDB-inhibitable Na-H exchange, Na influx across the luminal border of proximal colon was measured and was found to be inhibited equally by PDB and amiloride. We conclude that PDB, by activation of protein kinase C, inhibits electro-neutral amiloride-sensitive Na-H exchange in the luminal membrane of proximal colon.
Topics: Animals; Chlorides; Colon; Hydrogen; Ion Exchange; Male; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rabbits; Sodium
PubMed: 3864381
DOI: 10.1152/ajpcell.1985.249.5.C527 -
Experimental Cell Research Sep 1983The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and... (Comparative Study)
Comparative Study
The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and tumor-promoting activity were used. Treatment of cells with tumor-promoting phorbol esters resulted in accumulation of gangliosides and increased incorporation of [1-14C]palmitate and [9-3H]sialic acid into gangliosides. Moreover, the phorbol esters were found to increase the activity of CMP-sialic acid: lactosylceramide sialyltransferase, the enzyme catalysing the first step in ganglioside biosynthesis. The magnitude of phorbol ester effects on V79 cell ganglioside metabolism correlated with the in vivo phorbol ester tumor-promoting activity.
Topics: Animals; Carcinogens; Cell Division; Cell Line; Contact Inhibition; Cricetinae; Gangliosides; Palmitic Acid; Palmitic Acids; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Sialic Acids; Sialyltransferases; Tetradecanoylphorbol Acetate
PubMed: 6578051
DOI: 10.1016/0014-4827(83)90210-0 -
The American Journal of Physiology Jun 1986To determine whether protein kinase C is present in the basolateral membrane of the renal proximal tubular cell, we performed experiments to ascertain whether specific...
To determine whether protein kinase C is present in the basolateral membrane of the renal proximal tubular cell, we performed experiments to ascertain whether specific binding of [3H]phorbol 12,13-dibutyrate could be demonstrated in basolateral membranes isolated from canine kidney. Specific binding was demonstrable that was half maximal at between 10(-7) and 10(-8) M phorbol 12,13-dibutyrate. Binding was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other tumor-promoting phorbol esters, but not by inactive phorbol esters, including 4 alpha-phorbol. Incubation of basolateral membranes with TPA and phorbol 12,13-dibutyrate, but not with 4 alpha-phorbol, in the presence of submicromolar concentrations of free calcium, enhanced phosphorylation of several proteins demonstrable in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels originating from membranes subsequently exposed to [gamma-32P]ATP for 30 s. Dephosphorylation of [32P]phosphoproteins was observed in gels from membranes incubated with [gamma-32P]ATP over time. TPA-stimulated phosphorylation of one protein band with Mr 135,000 was quantitated and was found to increase as a function of [TPA]. Half-maximal TPA-stimulated phosphorylation of this protein band occurred at slightly less than 10(-9) M TPA. Our findings are consistent with a role for protein kinase C-effected phosphorylation of basolateral membrane proteins in the mediation or modulation of hormonal actions in the proximal tubular cell.
Topics: Adenosine Triphosphate; Amino Acids; Animals; Dogs; In Vitro Techniques; Kidney; Membranes; Microvilli; Osmolar Concentration; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phospholipids; Phosphorylation; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tritium; alpha-Glucosidases
PubMed: 3521324
DOI: 10.1152/ajprenal.1986.250.6.F1073 -
Cancer Research Oct 1983Biologically active phorbol ester derivatives displace [3H]phorbol-12, 13-dibutyrate from thioglycollate-elicited mouse peritoneal macrophages in a time-, temperature-,...
Biologically active phorbol ester derivatives displace [3H]phorbol-12, 13-dibutyrate from thioglycollate-elicited mouse peritoneal macrophages in a time-, temperature-, and concentration-dependent manner. Scatchard analysis revealed an apparent Kd of 54.1 nM and 8.0 X 10(5) sites/cell, indicating that these macrophages possess saturable, high-affinity phorbol ester-binding sites. These derivatives also act as chemoattractants for the macrophage at equivalent concentrations. A notable exception to this pattern is phorbol-12,13-diacetate. Phorbol-12,13-diacetate inhibits specific binding of [3H]phorbol-12,13-dibutyrate (concentration required for a 50% inhibition of the maximum specific binding of [3H]phorbol-12,13-dibutyrate, 2.6 microM) and chemotaxis to phorbol-12-myristate, 13-acetate (concentration required for a 50% inhibition of the maximum chemotactic response, 0.39 microM) while exhibiting no activity as a chemoattractant at concentrations up to 10(-5) M. The data indicate that phorbol-12,13-diacetate may be an antagonist for receptor-mediated chemotaxis to phorbol-12-myristate, 13-acetate in the macrophage.
Topics: Animals; Ascitic Fluid; Binding, Competitive; Caenorhabditis elegans Proteins; Carrier Proteins; Chemotaxis; Female; Macrophages; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug; Tetradecanoylphorbol Acetate; Time Factors
PubMed: 6309370
DOI: No ID Found -
Cancer Research Oct 1983The effect of phorbol dibutyrate (PDB) on the cell surface antigens of the human T-cell acute lymphoblastic leukemia cell line, Jurkat, was studied with OKT monoclonal...
The effect of phorbol dibutyrate (PDB) on the cell surface antigens of the human T-cell acute lymphoblastic leukemia cell line, Jurkat, was studied with OKT monoclonal antibodies by indirect immunofluorescence assay. Cells were analyzed in an Ortho Spectrum III fluorescence-activated flow cytometer. The surface antigen profile of untreated Jurkat cells resembled that of thymocytes; high levels of T3, T4, T6, T8, T9, T10, and T11 antigens were detected. Although 89% of cells were positive for T11, the putative sheep erythrocyte receptor, only 12% were able to form erythrocyte (sheep) rosettes. Exposure of the cells to 1.0 microM PDB for up to 7 days resulted in a rapid loss in T4 expression and a slower decrease in T6 reactivity, while the percentage of cells positive for T3, T8, T10, and T11 remained high. T4 reappeared on the cell surface when PDB was removed by washing. T11 antigen density increased 70%, and this was accompanied by an increase in the percentage of erythrocyte-rosetting cells from 12 to 55%. These changes in cell surface antigens induced by PDB suggested differentiation to a more mature state (i.e., a precursor cytotoxic-suppressor T-lymphocyte, T3+T8+T10+T11+). However, the reversibility of the change in T4 expression indicated that T4 loss was not a manifestation of terminal differentiation but rather was consistent with a phorbol ester-induced modulation of the cell surface T4 antigen.
Topics: Antigens, Surface; Cell Division; Cell Line; Humans; Leukemia, Lymphoid; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Rosette Formation; T-Lymphocytes
PubMed: 6603901
DOI: No ID Found -
Proceedings of the National Academy of... Oct 1988The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic...
The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3H]bryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structure-activity relations for the bryostatin class of compounds provide additional support for the validity of the model.
Topics: Animals; Brain; Bryostatins; Enzyme Activation; Female; Lactones; Macrolides; Mice; Models, Chemical; Phorbol Esters; Protein Kinase C; Structure-Activity Relationship
PubMed: 3174627
DOI: 10.1073/pnas.85.19.7197 -
The Journal of Biological Chemistry Aug 2010Protein kinase C (PKC) is considered crucial for hormonal Na(+)/H(+) exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we...
Protein kinase C (PKC) is considered crucial for hormonal Na(+)/H(+) exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we report that rather than PKC, direct binding of PEs/diacylglycerol to the NHE1 lipid-interacting domain (LID) and the subsequent tighter association of LID with the plasma membrane mainly underlies NHE1 activation. We show that (i) PEs directly interact with the LID of NHE1 in vitro, (ii) like PKC, green fluorescent protein (GFP)-labeled LID translocates to the plasma membrane in response to PEs and receptor agonists, (iii) LID mutations markedly inhibit these interactions and PE/receptor agonist-induced NHE1 activation, and (iv) PKC inhibitors ineffectively block NHE1 activation, except staurosporin, which itself inhibits NHE1 via LID. Thus, we propose a PKC-independent mechanism of NHE1 regulation via a PE-binding motif previously unrecognized.
Topics: Binding Sites; Cation Transport Proteins; Cell Line; Cell Membrane; Endocytosis; Humans; Phorbol Esters; Protein Kinase C; Receptors, Drug; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers
PubMed: 20551318
DOI: 10.1074/jbc.M110.130120 -
European Journal of Pharmacology Aug 1995This study investigates the relationship between the rate of phorbol ester-induced contraction of intact rat aorta and protein kinase C activation, as assessed by the...
This study investigates the relationship between the rate of phorbol ester-induced contraction of intact rat aorta and protein kinase C activation, as assessed by the translocation of protein kinase C from the cytosolic to the particulate fraction. Aorta was exposed to Ca(2+)-free physiologic salt solution prior to phorbol ester to prevent Ca(2+)-induced protein kinase C translocation during tissue homogenization. Phorbol myristate acetate, as well as phorbol dibutyrate, decreased cytosolic and/or increased particulate protein kinase C activity as early as 5 s following phorbol ester addition, which was prior to, or coincident with, the onset of contraction. These results suggests that phorbol ester-induced contraction of intact vascular smooth muscle is associated in a time-dependent manner with protein kinase C activation.
Topics: Animals; Aorta, Thoracic; Enzyme Activation; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Phorbol Esters; Protein Kinase C; Rats; Rats, Sprague-Dawley; Time Factors; Translocation, Genetic
PubMed: 7589220
DOI: 10.1016/0922-4106(95)90001-2 -
Cancer Research Oct 1987Bryostatin 1, a macrocyclic lactone, functions like the phorbol esters biochemically in binding to and activating protein kinase C. Biologically, however, although it...
Bryostatin 1, a macrocyclic lactone, functions like the phorbol esters biochemically in binding to and activating protein kinase C. Biologically, however, although it induces some phorbol ester responses such as mitogenesis in Swiss 3T3 cells, it paradoxically blocks the effects of the phorbol esters on differentiation in HL-60 promyelocytic leukemia cells and Friend erythroleukemia cells. Since the phorbol esters induce proliferation and terminal differentiation in distinct subpopulations of epidermal basal cells, we have now examined the action of bryostatin 1 in that system. Bryostatin 1 decreased epidermal growth factor binding and induced ornithine decarboxylase activity, the latter a marker of proliferation. The magnitude of the maximal induction of ornithine decarboxylase was less than for phorbol 12,13-dibutyrate. Bryostatin 1 only transiently caused the morphological change typical of phorbol ester treatment and did not induce transglutaminase or cornified envelope production, markers of the differentiative pathway. Combined treatment with bryostatin 1 and phorbol 12,13-dibutyrate gave similar results to treatment with bryostatin 1 alone, i.e., slight reduction to complete inhibition of phorbol ester action, depending on the response. The mechanism may reflect time dependent block of the protein kinase C pathway by bryostatin 1 in this system; although bryostatin 1 inhibited epidermal growth factor binding at short incubation times (1-2 h), by 4 h of incubation its inhibition was markedly reduced and it correspondingly blocked inhibition of epidermal growth factor binding by phorbol 12,13-dibutyrate. Since induction of terminal differentiation is proposed to be an essential component of phorbol ester mediated tumor promotion in skin, our findings suggest that bryostatin 1 may function as an inhibitor of phorbol ester promotion.
Topics: Animals; Bryostatins; Cells, Cultured; Cycloheximide; Dactinomycin; Enzyme Activation; Epidermal Growth Factor; Epidermis; Lactones; Macrolides; Mice; Ornithine Decarboxylase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transglutaminases
PubMed: 2888531
DOI: No ID Found