RESUMEN
BACKGROUND: Although organic extracts of gromwell (Lithospermum erythrorhizon) have been shown to promote wound healing, the wound healing effects of water extracts of gromwell (WG) that are commonly used in traditional remedies have not been elucidated. OBJECTIVE: We investigated whether WG promotes the migration and/or proliferation of cultured human keratinocytes (CHK) or dermal fibroblasts in parallel with increases in lipid synthesis during in vitro wound healing. METHODS: CHK or fibroblasts were treated with 1-1,000 µg/ml WG for up to 48 h following scratch wound formation. Cell migration was assessed by measuring coverage (in percent) from the wound margin, while cell proliferation and lipid synthesis were determined by [(3)H]thymidine incorporation into DNA fractions, and [(3)H]palmitate or [(3)H]serine incorporation into lipid fractions, respectively. RESULTS: Low-dose WG (1 µg/ml) enhanced the wound coverage for both CHK and fibroblasts at 24 h, while cell proliferation was not altered in either cell types. Synthesis of both total lipids and individual lipid classes, including phospholipids, sphingolipids and neutral lipids, were found to be increased at 24 h in CHK treated with 1 µg/ml WG; in similarly treated fibroblasts, only the syntheses of sphingolipids (such as ceramides and glucosylceramides), but not other lipid species, were significantly increased. In contrast, a higher dose of WG (10-1,000 µg/ml) did not enhance wound coverage, and 100 µg/ml WG neither altered cell proliferation nor lipid synthesis in both CHK and fibroblasts. CONCLUSION: Low-dose WG (1 µg/ml) enhances the migration of both CHK and fibroblasts with increased lipid synthesis in an in vitro wound scratch model.
Asunto(s)
Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Lithospermum/química , Extractos Vegetales/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Lípidos/biosíntesis , Masculino , Medicina Tradicional de Asia Oriental , Extractos Vegetales/administración & dosificación , Solventes/química , Factores de Tiempo , Agua/química , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Hydrolysis of glucosylceramides (GlcCer) by beta-glucocerebrosidase generates ceramides, critical components of the epidermal permeability barrier. Ceramides also are involved in the regulation of cellular proliferation and differentiation in a variety of cell types. Whereas most studies have focused on ceramides and their sphingoid base metabolites as growth inhibitors, GlcCer apparently acts oppositely (i.e., as a mitogen). To determine whether enhancement of GlcCer content stimulates epidermal mitogenesis, we examined the response of hairless mouse epidermis to alterations in endogenous and/or exogenous GlcCer. Topical applications of conduritol B epoxide, a specific irreversible inhibitor of beta-glucocerebrosidase, increased epidermal GlcCer levels twofold, an alteration localized largely to the basal, proliferative cell layer (fourfold increase); and stimulated epidermal proliferation (2.3-fold elevation in [3H]thymidine incorporation; P < or = 0.001), localized autoradiographically again to the basal layer, and resulting in epidermal hyperplasia. Intracutaneous administration of GlcCer (2.0 mg) also stimulated epidermal DNA synthesis, while simultaneous treatment with conduritol B epoxide plus GlcCer resulted in an additive increase in DNA synthesis. These increases in epidermal proliferation could not be attributed either to altered epidermal permeability barrier function, or to nonspecific irritant effects, as determined by four separate criteria. These results strongly suggest that GlcCer directly stimulates epidermal mitogenesis.
Asunto(s)
Células Epidérmicas , Glucosilceramidas/farmacología , Animales , División Celular/efectos de los fármacos , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/metabolismo , Inflamación/fisiopatología , Inositol/análogos & derivados , Inositol/farmacología , Masculino , Ratones , Esfingolípidos/metabolismoRESUMEN
The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited beta-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function.
Asunto(s)
Epidermis/química , Glucosilceramidas/farmacocinética , Piel/citología , Administración Tópica , Animales , Permeabilidad de la Membrana Celular , Ciclohexenos , Células Epidérmicas , Compuestos Epoxi/farmacología , Glucosidasas/antagonistas & inhibidores , Inositol/análogos & derivados , Inositol/farmacología , Masculino , Ratones , Ratones Pelados , Piel/química , Esfingolípidos/análisis , Esfingolípidos/farmacocinéticaRESUMEN
Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis.
Asunto(s)
Epidermis/metabolismo , Esfingolípidos/fisiología , Aciltransferasas/análisis , Animales , Colesterol/metabolismo , Epidermis/efectos de los fármacos , Epidermis/ultraestructura , Masculino , Ratones , Ratones Pelados , Permeabilidad , Serina C-Palmitoiltransferasa , Esfingolípidos/biosíntesis , beta-Alanina/análogos & derivados , beta-Alanina/farmacologíaRESUMEN
Hydrolysis of glucosylceramide by beta-glucocerebrosidase results in ceramide, a critical component of the intercellular lamellae that mediate the epidermal permeability barrier. A subset of type 2 Gaucher patients displays ichthyosiform skin abnormalities, as do transgenic Gaucher mice homozygous for a null allele. To investigate the relationship between glucocerebrosidase deficiency and epidermal permeability barrier function, we compared the stratum corneum (SC) ultrastructure, lipid content, and barrier function of Gaucher mice to carrier and normal mice, and to hairless mice treated topically with bromoconduritol B epoxide (BrCBE), an irreversible inhibitor of glucocerebrosidase. Both Gaucher mice and BrCBE-treated mice revealed abnormal, incompletely processed, lamellar body-derived sheets throughout the SC interstices, while transgenic carrier mice displayed normal bilayers. The SC of a severely affected type 2 Gaucher's disease infant revealed similarly abnormal ultrastructure. Furthermore, the Gaucher mice demonstrated markedly elevated transepidermal water loss (4.2 +/- 0.6 vs < 0.10 g/m2 per h). The electron-dense tracer, colloidal lanthanum, percolated between the incompletely processed lamellar body-derived sheets in the SC interstices of Gaucher mice only, demonstrating altered permeability barrier function. Gaucher and BrCBE-treated mice showed < 1% and < 5% of normal epidermal glucocerebrosidase activity, respectively, and the epidermis/SC of Gaucher mice demonstrated elevated glucosylceramide (5- to 10-fold), with diminished ceramide content. Thus, the skin changes observed in Gaucher mice and infants may result from the formation of incompetent intercellular lamellar bilayers due to a decreased hydrolysis of glucosylceramide to ceramide. Glucocerebrosidase therefore appears necessary for the generation of membranes of sufficient functional competence for epidermal barrier function.
Asunto(s)
Epidermis/enzimología , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/deficiencia , Animales , Ciclohexenos , Epidermis/metabolismo , Epidermis/ultraestructura , Enfermedad de Gaucher/patología , Humanos , Inositol/análogos & derivados , Inositol/farmacología , Ratones , Ratones Pelados , Ratones Transgénicos , Permeabilidad , Esfingolípidos/análisisRESUMEN
Preclinical studies have suggested that synergistic antitumor toxicity occurs when methotrexate (MTX) is administered prior to 5-fluorouracil (FUra). A protocol of sequenced, overlapping infusions of MTX and FUra was designed to achieve 5 microM MTX serum levels lasting 36 h and 1 to 5 microM FUra levels lasting 24 h, with leucovorin started at the end of the MTX infusion. Thirty-nine patients with metastatic neoplasms received a total of 127 treatment courses; two-thirds of the patients had received prior treatment with radiation therapy or chemotherapy; most of the latter treatment regimens included MTX or FUra. In three patients, the duration of FUra infusion was prolonged up to 72 h to determine the toxic limits of therapy. Blood samples were collected during treatment courses to estimate the half-lives and total-body clearances of MTX and FUra. The initial serum half-lives and total-body clearances of both MTX and FUra appeared within the range of reported normal values. The terminal half-life of MTX appeared less than previously reported values, and there appeared to be a substantial delay in achieving a FUra steady-state concentration; these two differences may have resulted from either the prolonged intervals of drug infusion or from metabolic interaction between the two drugs. During the 127 courses of treatment, nearly one-half of the patients experienced mild toxicity occurring after at least one treatment, but this toxicity was predominantly Grade I mucositis and/or diarrhea. Of the three patients who received extended intervals of FUra infusion, none was able to tolerate more than 48 h of FUra without developing mucositis. Thirty-four patients were evaluable for response; no one experienced a complete response, but 11 (32%) patients had either a partial or minimal response. Adenocarcinomas as a group, arising from the lung, gut, breast, and unknown site, appeared to respond best. Sequenced MTX-FUra infusion by this schedule is a generally well-tolerated regimen that deserves further clinical assessment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Fluorouracilo/sangre , Semivida , Humanos , Masculino , Metotrexato/administración & dosificación , Metotrexato/efectos adversos , Metotrexato/sangre , Persona de Mediana EdadRESUMEN
In addition to its anti-coagulant effect, heparin inhibits the growth of several types of cells. Recent studies suggest that heparin inhibition of proliferation of cultured human keratinocytes, results primarily from interaction with keratinocyte-generated, heparin-binding autocrine growth factors. In this study, we evaluated whether non-anticoagulant heparin analogs, and oligosaccharide fragments of heparin, retain the growth-inhibitory properties of whole heparin on human keratinocytes. Second-passage neonatal keratinocytes were grown in serum-free keratinocyte growth medium, and the effect of heparin analogs was studied in the absence of exogenous growth factors using keratinocyte-conditioned medium. Cell proliferation was assessed by measurement of both DNA content and [3H]-thymidine incorporation. The addition of heparin inhibited the conditioned medium-stimulated keratinocyte proliferation in a dose-dependent manner, with 80% inhibition at or above 10 micrograms/ml. Moreover, heparin was not toxic to keratinocytes (as detected by propidium-iodide fluorescence and by retention of normal protein synthetic rate) and it did not induce terminal differentiation (as measured by cornified envelope formation). Furthermore, heparin stimulated protein secretion by keratinocytes without altering rates of protein synthesis. The growth-inhibitory effects of heparin oligosaccharides were directly proportional to their chain length. The hexasaccharide unit represented the minimum requirement for inhibition, whereas decasaccharide units demonstrated nearly equivalent growth inhibition to native heparin. Finally, two non-anticoagulant heparin analogs were equipotent with heparin in inhibiting autocrine-induced keratinocyte growth. These studies show that the growth-inhibitory activities of heparin are independent of the anticoagulant effects and that decasaccharides contain the optimal oligosaccharide chain length for the antiproliferative effect in human keratinocytes.
Asunto(s)
Heparina/análogos & derivados , Heparina/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Anticoagulantes/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Humanos , Oligosacáridos/farmacología , Fragmentos de Péptidos/farmacologíaRESUMEN
Omega-hydroxyceramides (omega-OHCer) are the predominant lipid species of the corneocyte lipid envelope in the epidermis. Moreover, their omega-esterified-derivatives (acylCer) are major components of the stratum corneum extracellular lamellae, which regulate cutaneous permeability barrier function. Because epidermal omega-OHCer appear to be generated by a cytochrome P450-dependent process, we determined the effects of a mechanism-based inhibitor of omega-hydroxylation, aminobenzotriazole (ABT), on epidermal omega-OH Cer formation and barrier function. We first ascertained that ABT, but not hydroxybenzotriazole (OHBT), a chemical relative with no P450 inhibitory activity, inhibited the incorporation of [14C]-acetate into the omega-OH-containing Cer species in cultured human keratinocytes (68.1% +/- 6.9% inhibition versus vehicle-treated controls; p < 0.001), without altering the synthesis of other Cer and fatty acid species. In addition, ABT significantly inhibited the omega-hydroxylation of very long-chain fatty acids in cultured human keratinocytes. Topical application of ABT, but not OHBT, when applied to the skin of hairless mice following acute barrier disruption by tape-stripping, resulted in a significant delay in barrier recovery (e.g., 38.3% delay at 6 h versus vehicle-treated animals), assessed as increased transepidermal water loss. The ABT-induced barrier abnormality was associated with: (i) a significant decrease in the quantities of omega-OHCer in both the unbound and the covalently bound Cer pools; (ii) marked alterations of lamellar body structure and contents; and (iii) abnormal stratum corneum extracellular lamellar membrane structures, with no signs of cellular toxicity. Furthermore, pyridine-extraction of ABT- versus vehicle-treated skin, which removes all of the extracellular lamellae, leaving the covalently attached lipids, showed numerous foci with absent corneocyte lipid envelope in ABT- versus vehicle-treated stratum corneum. These results provide the first direct evidence for the importance of omega-OHCer for epidermal permeability function, and suggest further that acylCer and/or corneocyte lipid envelope are required elements in permeability barrier homeostasis.
Asunto(s)
Ceramidas/fisiología , Epidermis/fisiología , Queratinocitos/fisiología , Lípidos/fisiología , Proteínas de la Membrana/fisiología , Administración Tópica , Animales , Ceramidas/antagonistas & inhibidores , Ceramidas/metabolismo , Células Epidérmicas , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Hidroxilación/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Pelados , Membrana Nuclear/fisiología , Orgánulos/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Valores de Referencia , Triazoles/farmacologíaRESUMEN
Human keratinocytes differentiate in vitro in response to a variety of stimuli, but neither the levels nor the spectrum of ceramides approach those seen in vivo. Ceramide production increases when human keratinocytes are grown at an air-liquid interface, and alterations in ceramide content occur when vitamin C is added to air-exposed, organotypic culture systems (Ponec et al. J Invest Dermatol 109:348, 1997). Here, we assessed whether vitamin C stimulates sphingolipid production in human keratinocytes independent of differentiation and air exposure. When submerged, human keratinocytes were grown in 1.2 mM calcium and serum-containing medium with vitamin C (50 microg per ml) for 9 d, total lipid content remained unchanged, but both glucosylceramide and ceramide content increased. Moreover, selected ceramide and glucosylceramide species: i.e., nonhydroxy ceramide 2 and both alpha- and omega-hydroxylated sphingolipids, increased preferentially [ceramide 4 (6-hydroxy-acylceramide), ceramide 5 (alpha-hydroxyceramide), ceramide 6 (4-hydroxy-alpha-hydroxyceramide), and ceramide 7 (6-hydroxy-alpha-hydroxyceramide); and acylglucosylceramide, glucosylceramide-B, and glucosylceramide-D], whereas ceramide 1, ceramide 3, glucosylceramide-C, and sphingomyelin remained unchanged. Synthesis of the corresponding ceramide and glucosylceramide fractions was enhanced by vitamin C, attributable, in part, to increased ceramide synthase activity (over 2-fold, p = 0.01); both serine palmitoyltransferase and glucosylceramide synthase activities remained unaltered. Finally, increased vitamin C-stimulated sphingolipid production correlated with the presence of lamellar bodies with mature internal contents, an increase in covalently bound omega-hydroxyceramide, and the appearance of prominent, corneocyte-bound lipid envelopes, whereas cornified envelope formation was unchanged. Thus, in submerged human keratinocytes, vitamin C induces both increased sphingolipid production and enhancement of permeability barrier structural markers.
Asunto(s)
Ácido Ascórbico/farmacología , Queratinocitos/metabolismo , Esfingolípidos/biosíntesis , Biomarcadores , Células Cultivadas , Ceramidas/metabolismo , Glucosilceramidas/metabolismo , Humanos , Inmersión , Queratinocitos/fisiología , Oxidorreductasas/metabolismoRESUMEN
Ultraviolet (UV) irradiation of the skin induces a variety of responses in the epidermis, including sunburn cell formation, epidermal hyperplasia, and a dose-dependent permeability barrier abnormality, an effect that appears to be dependent upon both UVB-induced hyperplasia and T-cell activation. Since intrinsically aged epidermis displays decreased epidermal turnover, diminished inflammatory response to various stimuli, including UVR, and impaired immune function, we investigated the effects of UVB on both epidermal barrier function and proliferation in hairless mice of increasing chronologic age (27, 61, and 90 wk). After a single UVB exposure (0.15 J/cm2 7.5 MED), a barrier abnormality developed (i.e., increased transepidermal water loss; TEWL), after a delay of > or = 48 h, regardless of age. In young mice (27 wk old), TEWL levels peaked at 72-96 h (9.9-fold over untreated controls), whereas increased epidermal [3H]thymidine incorporation preceded the peak TEWL increase (i.e., approximately 570% increase over controls at 48 h). In contrast, the UVB-induced increased in both TEWL and DNA synthesis were significantly diminished, with decreased epidermal hyperplasia evident, in intrinsically aged versus young mouse epidermis. Baseline epidermal thickness decreased with animal age (i.e., 16.8 +/- 3.1 vs. 27.9 +/- 0.7 microm for 90- vs. 27-wk-old animals, respectively; p < 0.02), suggesting that the diminished barrier response with aging reflects an attenuation of events subsequent to initial UVB exposure, rather than an increase in the UV dose delivered. These results demonstrate that (i) murine epidermis becomes less sensitive to UVB-induced barrier alterations with age and (ii) decreased DNA synthesis after UVB correlates with the age-related decrease in barrier dysfunction.
Asunto(s)
División Celular/fisiología , División Celular/efectos de la radiación , Permeabilidad de la Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de la radiación , Envejecimiento de la Piel/efectos de la radiación , Rayos Ultravioleta , Animales , ADN/biosíntesis , Masculino , Ratones , Ratones Pelados , Grosor de los Pliegues Cutáneos , Linfocitos T/citologíaRESUMEN
Glucosylceramides (GlcCer) and ceramides (Cer) appear to have opposite effects on epidermal growth and differentiation. Whereas Cer inhibit mitosis and induce terminal differentiation and apoptosis in cultured keratinocytes, GlcCer is mitogenic in young murine epidermis. Using a recently described murine model of chronologic senescence we explored whether GlcCer is mitogenic in aged epidermis. Epidermal GlcCer content increases following topical applications of either conduritol-B epoxide (CBE), an inhibitor of GlcCer hydrolysis, or exogenous GlcCer in a penetration-enhancing vehicle. During chronologic aging in the hairless mouse, baseline epidermal DNA synthesis rates remain normal until 18 mo, but decline significantly at 24 mo. Topical CBE stimulates a 1.5- to 1.9-fold increase in epidermal DNA synthesis in all age groups (i.e., 1-2, 18, and 24 mo). Although the CBE induced increase in [3H]thymidine incorporation in 24 mo old animals is significant (p < 0.01), it is not sufficient to reach the absolute levels reached in similarly treated, younger mouse epidermis. Moreover, topical GlcCer induced mitogenesis is both dose dependent and hexose specific in young (1-2 mo old) animals, and remains effective in aged (< or = 24 mo old) animals. Furthermore, the CBE induced increase in DNA synthesis in aged epidermis is sufficient to produce epidermal hyperplasia. Finally, although an increased GlcCer:Cer ratio can alter stratum corneum barrier function and membrane structure, neither stratum corneum function nor extracellular membrane structure change under these experimental conditions, and therefore the mitogenic effects of increased epidermal GlcCer cannot be attributed to effects on the stratum corneum. These results show that: (i) elevations in endogenous GlcCer are mitogenic for aged as well as young murine epidermis; (ii) topical GlcCer is also mitogenic when delivered in an enhancing vehicle; and (iii) despite the putative importance of epidermal DNA synthesis for barrier homeostasis, these mitogenic alterations do not alter stratum corneum function.
Asunto(s)
Envejecimiento/fisiología , Epidermis/efectos de los fármacos , Glucosilceramidas/farmacología , Mitógenos/farmacología , Administración Tópica , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/fisiología , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Células Epidérmicas , Epidermis/patología , Glucosilceramidasa/antagonistas & inhibidores , Hiperplasia , Inositol/análogos & derivados , Inositol/farmacología , Masculino , Ratones , Ratones PeladosRESUMEN
Prior studies have established the requirement for enzymatic hydrolysis of glucosylceramides to ceramide for epidermal barrier homeostasis. In this study, we asked whether sphingomyelin-derived ceramide, resulting from acid-sphingomyelinase activity, is also required for normal barrier function. We showed first, that a subset of Niemann-Pick patients with severe acid-sphingomyelinase deficiency (i.e., <2% residual activity) demonstrate abnormal permeability barrier homeostasis, i.e., delayed recovery kinetics following acute barrier disruption by cellophane tape-stripping. To obtain further mechanistic insights into the potential requirement for sphingomyelin-to-ceramide processing for the barrier, we next studied the role of acid-sphingomyelinase in hairless mouse skin. Murine epidermis contains abundant acid-sphingomyelinase activity (optimal pH 5.1-5.6). Two hours following acute barrier disruption by tape-stripping, acid-sphingomyelinase activity increases 1. 44-fold (p<0.008 versus vehicle-treated controls), an increase that is blocked by a single topical application of the acid-sphingomyelinase inhibitor, palmitoyldihydrosphingosine. Furthermore, both palmitoyldihydrosphingosine and desipramine, a chemically and mechanically unrelated acid-sphingomyelinase inhibitor, significantly delay barrier recovery both 2 and 4 h after acute barrier abrogation. Inhibitor application also causes both an increase in sphingomyelin content, and a reduction of normal extracellular lamellar membrane structures, in the stratum corneum. Both of the inhibitor-induced delays in barrier recovery can be overridden by co-applications of topical ceramide, demonstrating that an alteration of the ceramide-sphingomyelin ratio, rather than sphingomyelin accumulation, is likely responsible for the barrier abnormalities that occur with acid-sphingomyelinase deficiency. These studies demonstrate an important role for enzymatic processing of sphingomyelin-to-ceramide by acid-sphingomyelinase as a mechanism for generating a portion of the stratum corneum ceramides for permeability barrier homeostasis in mammalian skin.
Asunto(s)
Permeabilidad de la Membrana Celular , Ceramidas/fisiología , Enfermedades de Niemann-Pick/fisiopatología , Esfingomielinas/fisiología , Adolescente , Adulto , Permeabilidad de la Membrana Celular/fisiología , Niño , Femenino , Homeostasis/fisiología , Humanos , Masculino , Persona de Mediana Edad , Piel/citología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
UV irradiation induces a variety of cutaneous responses, including disruption of epidermal permeability barrier function, the basis for which is not known. Herein, we investigated the separate roles of hyperproliferation and inflammation in the pathogenesis of UVB-induced barrier disruption. Adult hairless mice were exposed to increasing doses of UVB (1.5-7.5 MED), and transepidermal water loss (TEWL) was monitored daily for up to 7 d. The extent of TEWL increase was dependent on the UVB dose, but with all doses, the increase began after > or =48 h and peaked at 96 h, decreasing by 120 h. Epidermal [(3)H]thymidine incorporation increased at 24 h and peaked at 48 h (570%), preceding the maximal increase in TEWL. Cyclosporin A, methotrexate, 5-fluorouracil, or arabinosylcytosine significantly diminished the UVB-induced TEWL increase. Athymic nude mice also displayed a markedly diminished response to UVB, and DNA synthesis did not increased at 48 h. Transplantation of athymic mice with T-cell-enriched mixed immune cells significantly restored sensitivity to both the UVB-induced hyperproliferation and the barrier defect. Finally, although UVB exposure increased PGE2 levels in whole skin samples (2- to 3-fold within 1-3 h; p < 0.005), this increase was completely blocked by topical indomethacin, and neither topical indomethacin nor topical glucocorticoids blocked development of the barrier abnormality. These results show that (i) UVB produces delayed alteration in barrier function and (ii) both an epidermal proliferative response and thymocyte-mediated events (but not PGE2 production and nonspecific inflammation) appear to contribute to UVB-induced abrogation of the permeability barrier.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de la radiación , Rayos Ultravioleta , Corticoesteroides/fisiología , Animales , División Celular , ADN/biosíntesis , Dinoprostona/biosíntesis , Relación Dosis-Respuesta en la Radiación , Hiperplasia , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos BALB C , Ratones Desnudos , Piel/patología , Piel/efectos de la radiación , Timo/citologíaRESUMEN
The extracellular lipids of the stratum corneum, which are comprised mainly of cholesterol, fatty acids, and ceramides, are essential for epidermal permeability barrier function. Moreover, disruption of the permeability barrier results in an increased cholesterol, fatty acid, and ceramide synthesis in the underlying epidermis. This increase in lipid synthesis has been shown previously to be due to increased activities of HMG-CoA reductase, acetyl-CoA carboxylase, fatty acid synthase and serine palmitoyl transferase, key enzymes of cholesterol, fatty acid, and ceramide synthesis, respectively. In the present study, we determined whether the mRNA levels for the key enzymes required for synthesis of these three classes of lipids increase coordinately during barrier recovery. By northern blotting, the steady-state mRNA levels for HMG-CoA reductase, HMG-CoA synthase, farnesyl pyrophosphate synthase, and squalene synthase, key enzymes for cholesterol synthesis, all increased significantly after barrier disruption by either acetone or tape stripping. Additionally, the steady-state mRNA levels of acetyl-CoA carboxylase and fatty acid synthase, required for fatty acid synthesis, as well as serine palmitoyl transferase, the rate-limiting enzyme of de novo ceramide synthesis, also increased. Furthermore, artificial restoration of the permeability barrier by occlusion after barrier disruption prevented the increase in mRNA levels for all of these enzymes, except farnesyl pyrophosphate synthase, indicating a specific link of the increase in mRNA levels to barrier requirements. The parallel increase in epidermal mRNA levels for the enzymes required for cholesterol, fatty acid, and ceramide synthesis may be due to one or more transcription factors that regulate lipid requirements for permeability barrier function in keratinocytes.
Asunto(s)
Ceramidas/biosíntesis , Colesterol/biosíntesis , Epidermis/metabolismo , Ácidos Grasos/biosíntesis , ARN Mensajero/análisis , Aciltransferasas/genética , Animales , Ácido Graso Sintasas/genética , Hidroximetilglutaril-CoA Reductasas/genética , Masculino , Ratones , Ratones Pelados , Permeabilidad , Serina C-PalmitoiltransferasaRESUMEN
Leukemia cells isolated from eight patients with acute leukemia before treatment were examined for in vitro uptake of daunorubicin (DNR) and inhibition of DNA synthesis. In addition, plasma and cellular levels of DNR and daunorubicinol (DOL) were examined in six of the eight patients. Inhibition of DNA synthesis was determined with a 3H-thymidine incorporation assay. In vitro cellular 14C-DNR was quantified by means of liquid scintillation spectrometry, whereas in vivo DNR and DOL concentrations were determined by high-performance liquid chromatography. In vitro intracellular plateau concentrations of DNR were achieved within 1-2 h after continuous exposure to 0.01, 0.1, and 1.0 microgram/ml in the majority of cases. Based on our in vitro studies, a dose-response curve was found between increasing intracellular DNR and incorporation of 3H-thymidine. Peak intracellular levels of DNR after treatment occurred immediately after administration of the drug, whereas intracellular DOL levels accumulated over several hours. Plasma concentrations of DNR and DOL were not useful in estimating target tissue concentrations or inhibition of 3H-thymidine incorporation. Extrapolation of in vivo cellular DNR concentrations to the in vitro dose-response curve allows an estimate of DNR sensitivity.
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ADN de Neoplasias/biosíntesis , Daunorrubicina/metabolismo , Leucemia/tratamiento farmacológico , Adolescente , Adulto , Anciano , Transporte Biológico , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Daunorrubicina/análogos & derivados , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The pharmacokinetic behavior of carboplatin administered by the i.p. route at a dose of 200 mg/m2 was studied during five courses of therapy in four patients with ovarian cancer. A regional pharmacologic advantage was noted with carboplatin administered by this route, with peak peritoneal fluid concentrations 18-fold those in plasma, and area under the curve (AUC) for the peritoneum showing a 18-fold and 6-fold increase over plasma AUC at 4 and 24 h, respectively. The mean residence time of total platinum in the peritoneum was 4.7 h. Approximately 10% and 40% of plasma platinum was protein bound at 4 and 24 h after treatment, respectively, whereas peritoneal fluid platinum showed minimal protein binding. Peak plasma platinum levels were comparable to those recorded in previous studies with i.v. doses of carboplatin. Peritoneal clearance of carboplatin in these four patients appeared to be less than that previously reported for cisplatin. Further studies are in progress with higher doses of i.p. carboplatin.
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Antineoplásicos/administración & dosificación , Carcinoma/tratamiento farmacológico , Compuestos Organoplatinos/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Líquido Ascítico/metabolismo , Proteínas Sanguíneas/metabolismo , Carboplatino , Evaluación de Medicamentos , Femenino , Humanos , Infusiones Parenterales , Cinética , Compuestos Organoplatinos/sangre , Compuestos Organoplatinos/metabolismo , Unión Proteica , Factores de TiempoRESUMEN
The purpose of this study was to determine whether changes in cellular lipid composition accompanied the selection of cells that are resistant to the anthracycline doxorubicin. Total cellular lipid extracts from doxorubicin-sensitive and doxorubicin-resistant P388 murine leukemia cells were prepared and separated into neutral glycosphingolipids, gangliosides, phospholipids, and neutral lipid families. No significant quantitative differences in total cholesterol, lipid-bound sialic acid, neutral hexose, and lipid-bound phosphate were found between the two cell lines. Gas-liquid chromatographic analysis of the fatty acids derived from each lipid class demonstrated that sensitive and resistant cells had essentially identical fatty acid compositions. Qualitative evaluation of the four lipid classes by high-performance thin-layer chromatography revealed only minor differences in lipid composition between the resistant and the sensitive cells. Results from this study indicate that although minor differences between the two cell lines are present, no major cellular lipid differences are evident to account for the marked differences in the cellular pharmacokinetics and cytotoxic effects of doxorubicin between doxorubicin-sensitive and doxorubicin-resistant P388 murine leukemia a cells.
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Doxorrubicina/farmacología , Leucemia P388/metabolismo , Leucemia Experimental/metabolismo , Lípidos de la Membrana/metabolismo , Animales , Cromatografía de Gases , Cromatografía en Capa Delgada , Resistencia a Medicamentos , Leucemia P388/tratamiento farmacológico , RatonesRESUMEN
Diethyldithiocarbamate (DDTC), a chelating agent that is a major metabolite of disulfuram, has been proposed as a potential rescue agent to reduce toxicity following high-dose cisplatin (HDCP) therapy. In the present study, we examined the pharmacologic interaction of HDCP and DDTC given as rescue therapy. Total plasma platinum and ultrafiltrate platinum pharmacokinetics and DDTC levels were determined in six patients with advanced malignancies who received a total of 11 cycles of HDCP with DDTC rescue. HDCP therapy (200 mg/m2 per cycle) consisted of 100 mg/m2 reconstituted in 250 cc 3% saline and infused over 3 h on days 1 and 8 of each 28-day cycle. DDTC rescue at a dose of 4 gm/m2 was given by an i.v. infusion (duration 1.5-3.5 h), beginning 45 min after the completion of cisplatin infusion. Peak total and ultrafiltrate levels and cisplatin pharmacokinetics in this study were indistinguishable from those of previous studies using the same HDCP regimen without DDTC rescue. Ultrafiltrate or unbound plasma platinum was less than 10% of total plasma platinum concentrations and demonstrated a biphasic pattern of elimination. Levels of DDTC predicted to be chemoprotective (greater than 400 microM) were achieved with the dose and schedule used in this study. These data demonstrate that DDTC can be targeted to protective plasma concentrations without significantly altering plasma cisplatin pharmacokinetics and support the potential usefulness of DDTC as a rescue agent following HDCP therapy.
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Cisplatino/administración & dosificación , Ditiocarba/administración & dosificación , Cisplatino/efectos adversos , Cisplatino/sangre , Cisplatino/farmacocinética , Ditiocarba/sangre , Ditiocarba/farmacocinética , Evaluación de Medicamentos , Interacciones Farmacológicas , Humanos , Espectrofotometría Atómica , Factores de TiempoRESUMEN
Sphingolipids, the predominant lipid species in mammalian stratum corneum play, a central role in permeability barrier homeostatis. Prior studies have shown that the epidermis synthesizes abundant sphingolipids, a process regulated by barrier requirements, and that inhibition of sphingolipid synthesis interferes with barrier homeostasis. To investigate further the relationship between epidermal sphingolipid metabolism and barrier function, we localized sphingolipid synthetic activity in murine epidermis under basal conditions, and following acute (acetone treatment) or chronic (essential fatty acid deficiency, EFAD) barrier perturbation, using dithiothreitol and/or the staphylococcal epidermolytic toxin to isolate the lower from the outer epidermis. Under basal conditions, both the activity of serine palmitoyl transferase (SPT), the rate-limiting enzyme of sphingolipid synthesis, and the rates of 3H-H2O incorporation into sphingolipids were nearly equivalent in the lower and the outer epidermis. Following acute barrier perturbation, SPT activity increased significantly in both the lower (35%; P < 0.05) and the outer epidermal layers (60%; P < 0.01). The rates of 3H-H2O incorporation into each major sphingolipid family, including ceramides, glucosylceramides and sphingomyelin, increased significantly in both the lower and the outer epidermis of treated flanks after acute barrier disruption. Finally, SPT activity was modestly elevated (20%; P < 0.01) in the lower but not in the outer epidermis of EFAD animals. These studies demonstrate the ability of both lower and outer epidermal cells to generate sphingolipids, and that permeability barrier homeostatic mechanisms appear to differentially regulate SPT activity and sphingolipid synthesis in the lower and the outer epidermis in response to acute and chronic barrier perturbation.(ABSTRACT TRUNCATED AT 250 WORDS)
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Aciltransferasas/metabolismo , Epidermis/metabolismo , Esfingolípidos/biosíntesis , Acetona/toxicidad , Animales , Colesterol/biosíntesis , Epidermis/efectos de los fármacos , Ácidos Grasos Esenciales/deficiencia , Homeostasis , Masculino , Ratones , Ratones Pelados , Permeabilidad/efectos de los fármacos , Serina C-PalmitoiltransferasaRESUMEN
Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases which play key roles in extracellular matrix remodeling, connective tissue damage, inflammation and cell proliferation in a variety of tissues. Since MMP inhibitors have been recently shown to decrease proliferation of vascular smooth-muscle cells, and to prevent neutrophil infiltration in response to alkali burns, we sought to determine whether MMPs play a role in the pathogenesis of inflammatory or hyperproliferative skin disorders. The effects of a specific MMP inhibitor and its analogues on phorbol dibutyrate (PdiBu)-induced inflammation and epidermal hyperplasia in murine skin were assessed. Topical GM 6001, a hydroxamic acid analog with potent inhibitory activity against several MMPs, markedly inhibited PdiBu-induced increases in both ear thickness and ear punch-biopsy weight in a dose-dependent manner 30 h after topical application of PdiBu. Maximal inhibition (75%) was obtained at a dose of 100 micrograms/cm2 (P < 0.01). Moreover, histologic analysis revealed that GM 6001 decreased both the inflammatory cellular infiltrates and epidermal hyperplasia induced by PdiBu. Whereas similar results were found for GM 1489, an analog of GM 6001, acetohydroxamic acid, containing the critical metal ligand group but without the amino acid side chains necessary for binding to the MMPs, did not alter the response to PdiBu inflammation/hyperplasia. These results show that the MMP inhibitors, GM 6001 and GM 1489, are effective in reducing both the inflammatory and hyperproliferative responses that occur following topical phorbol ester application, suggesting a potential role for MMPs in cutaneous inflammatory dermatoses. Moreover, the delivery of this class of inhibitors across intact stratum corneum implies that MMP inhibition could provide an approach to the topical treatment of inflammatory dermatoses.