Application Of Phorbol 12-myristate 13-acetate Research Articles

Effect of resiniferatoxin pretreatment on the inflammatory response to phorbol-12-myristate-13-acetate in mouse strains with different susceptibilities to phorbol ester tumor promotion

All tumor-promoting phorbol esters induce inflammation in mouse skin. The correlation between promoting and inflammatory activities is only partial, however, indicating that only some events in inflammation may be closely coupled to the process of tumor promotion. Resiniferatoxin (RTX), an extremely inflammatory phorbol-related diterpene, acts as an ultrapotent analog of capsaicin to stimulate and then to block the neurogenic inflammatory pathway. In CD-1 mice, we have used pretreatment with RTX to show that the erythema and edema responses to phorbol and 12-deoxyphorbol esters in significant part involve this neurogenic inflammatory pathway. We report here that mouse strains with differing sensitivities to phorbol-ester-induced promotion displayed marked differences in the effect of pretreating with RTX on the edema response following phorbol-12-myristate-13-acetate (PMA) application. In the highly promotion-sensitive SENCAR mouse, RTX pretreatment had little inhibitory effect; the edema response to PMA was similar with or without RTX pretreatment 6 h before PMA application. On the other hand, in C57BL/6J mice, which are resistant to promotion by phorbol esters under the usual protocols, the edema response to PMA was totally eliminated by RTX pretreatment during the first 8 h after PMA administration. DBA/2J mice, which are similar to CD-1 mice in their susceptibility to PMA promotion, responded similarly to CD-1: the edema response was blocked partially by RTX pretreatment during the early phase (up to 8 h) of inflammation. Our results suggest that the RTX-resistant component of PMA-induced edema may correlate better with the sensitivity to promoting action than does the overall inflammatory response.

Phorbol esters stimulate macropinocytosis and solute flow through macrophages.

The morphology and kinetics of pinocytosis by bone marrow-derived macrophages were studied to determine how stimulation by phorbol esters increases net solute accumulation. Application of phorbol myristate acetate (PMA) increased both the abundance of macropinosomes and the rate of solute flow through the endocytic compartment. The large pinosomes originated as ruffles at the cell margins that folded back on themselves, internalizing extracellular medium and solutes. I examined how stimulation affects the kinetics of pinocytic influx, accumulation, and subsequent efflux of the fluorescent dye Lucifer Yellow (LY) in macrophages. Both the accumulation of LY and its subsequent efflux were temperature-dependent and directly proportional to the concentration of LY in the extracellular medium. Macrophages incubated in PMA and LY for 2 h accumulated four to six times more LY than did macrophages in LY alone. If after pinocytosis the macrophages were washed and reincubated in unlabeled medium for a 1 h chase period, some of the internalized LY was regurgitated from the cells. Inclusion of PMA in the chase medium increased efflux of LY. In contrast, a smaller percentage of LY was regurgitated from macrophages which were both loaded and chased in the presence of PMA. This indicates that although efflux is increased by PMA, influx increases more, and therefore more of the LY entering by pinocytosis is retained within the cell. I suggest that macropinocytosis increases the size difference between pinosomes and efflux vesicles, and that that difference increases greatly both solute accumulation and membrane flow through the endocytic compartment.

Insulin and phorbol ester stimulate conductive Na+ transport through a common pathway.

Insulin stimulates Na+ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na+ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na+ transport across frog skin. In the present work, we have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na+ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. Preincubation with D-sphingosine, an inhibitor of protein kinase C, also reduces the natriferic action of insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na+ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.

Evaluation of granule exocytosis in toad urinary bladder.

Addition of phorbol myristate acetate (PMA) to the mucosal bathing solution induces exocytosis of the granules found in the apical cytoplasm of toad urinary bladder epithelial cells. Because mucosal application of PMA also increases epithelial water permeability in the complete absence of vasopressin, these observations have suggested that granules might have a role in mediating the permeability response to vasopressin. From electron microscopic immunolocalization studies we have identified a 70-kDa protein as a component of the apical surface glycocalyx that is stored in the granules. Using antibodies to this protein in a competitive immunoassay, we found that addition of PMA to the mucosal side results in more than a fourfold increase in antigen release into the mucosal bath. The amount of antigen detectable on the mucosal surface of the cells was doubled by mucosal PMA exposure. Serosal application of PMA or addition of the inactive analogue phorbol didecanoate to either surface failed to produce significant antigen release. These results are consistent with electron microscopic studies showing exocytosis of granules only with mucosal exposure to PMA. However, immunoassay of granule antigen after vasopressin stimulation of water permeability failed to show a detectable change in granule exocytosis. We conclude that granule exocytosis does not play an important role in mediating vasopressin-induced changes in permeability.

Increase in histidine decarboxylase activity in skin of genetically mast-cell-deficient W/Wv mice after application of phorbol 12-myristate 13-acetate: evidence for the presence of histamine-producing cells without basophilic granules.

Histidine decarboxylase (HisDCase, EC 4.1.1.22) activity in mouse skin increased by a factor of more than 10 after a single application of phorbol 12-myristate 13-acetate. The cell type that was responsible for the increase in HisDCase activity was examined by using (WB X C57BL/6)F1-W/Wv mice, which are genetically deficient in tissue mast cells. In contrast to a report that increase of ornithine decarboxylase (EC 4.1.1.17) activity occurs in the epidermis [O'Brien, T. G., Simisiman, R. C. & Boutwell, R. K. (1975) Cancer Res. 35, 2426-2433], the HisDCase activity was found to increase in the dermis. Although most of the histamine in the dermis was present in mast cells, the increase in HisDCase activity in the skin in W/Wv mice was comparable with that in congeneic +/+ mice. This increase of HisDCase activity in the skin of W/Wv mice was abolished by prior x-ray irradiation (800 rads; 1 rad = 0.01 gray) but was restored by subsequent bone marrow transplantation. Because mice, in general, are known to lack basophilic leukocytes, the present results suggest the existence of histamine-producing cells without basophilic granules that are derived from the bone marrow.

Initiating Activity of Aromatic Hydrocarbons in Two-Stage Carcinogenesis<xref ref-type="fn" rid="FN2">2</xref>

Compounds which are noncarcinogenic or weakly carcinogenic in most biological test systems have shown pronounced tumor-initiating activity on mouse skin. In these experiments, a single dose of the initiating agent was applied on mouse skin (ICR/Ha Swiss) followed by repeated application of phorbol myristate acetate for 1 year or longer. The classical “noncarcinogenic” aromatic hydrocarbon dibenz[a,c]anthracene, when tested in this manner, resulted in 25/70 mice with papillomas and 6/70 mice with squamous carcinoma of the skin after 1 year on test. When tested as an initiating agent, another weak carcinogen, benz[a]anthracene, also resulted in significant tumor yields: 10/20 mice with benign tumors. First tumors appeared at 51—74 days from the beginning of promoting treatment. The significance of these findings to tobacco carcinogenesis and other relevant aspects are discussed.