RESUMEN
A series of sulfonimidamide analogs of the oncolytic diarylsulfonylureas was synthesized and evaluated for (1) in vitro cytotoxicity against CEM cells, (2) in vivo antitumor activity against subaxillary implanted 6C3HED lymphosarcoma, and (3) metabolic breakdown to the o-sulfate of p-chloroaniline. The separated enantiomers of one sulfonimidamide analog displayed very different activities in the in vivo screening model. In general, several analogs demonstrated excellent growth inhibitory activity in the 6C3HED model when dosed orally or intraperitoneally. A correlative structure-activity relationship to the oncolytic sulfonylureas was not apparent.
Asunto(s)
Antineoplásicos/farmacología , Compuestos de Fenilurea/farmacología , Compuestos de Sulfonilurea/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , División Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C3H , Estructura Molecular , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/metabolismo , Sarcoma Experimental/tratamiento farmacológico , Compuestos de Sulfonilurea/síntesis química , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/metabolismo , Células Tumorales CultivadasRESUMEN
The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.
Asunto(s)
Antineoplásicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , Genes ras , Proteínas HSP70 de Choque Térmico/genética , Compuestos de Sulfonilurea/farmacología , Animales , Calcio/metabolismo , Línea Celular , Ácido Egtácico/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiología , Ratas , TransfecciónRESUMEN
Gemcitabine is a new deoxycytidine analog that exhibits significant cytotoxicity against a variety of cultured murine and human tumor cells. The cytotoxic action of gemcitabine appears to be due to the inhibition of DNA synthesis by inhibition of ribonucleotide reductase and by competition with dCTP for incorporation into DNA. We have previously shown that gemcitabine, but not cytosine arabinoside (ara-C), has a broad spectrum of antitumor activity against 7 different types of murine solid tumors. The activity of gemcitabine was schedule dependent. To further characterize its activity, gemcitabine was tested against 12 human carcinoma xenografts. When given on an every 3 day x 4 schedule, the following percent inhibitions (at maximally tolerated doses [MTD]; MTD/2) in tumor growth were seen: MX-1 mammary (93%; 80%), CX-1 colon (92%; 82%), HC-1 colon (96%; 92%), GC3 colon (98%; 94%), VRC5 colon (99%; 100%), LX-1 lung (76%; 61%), CALU-6 lung (75%; 38%), NCI-H460 lung (45%; 46%), HS766T pancreatic (73%; not tested), PaCa-2 pancreatic (69%; 40%), PANC-1 pancreatic (70%; 60%), and BxPC-3 pancreatic (9%; 19%). In contrast, only the LX-1 lung carcinoma xenograft was responsive to ara-C treatment, which inhibited tumor growth by a marginal 62 percent. Thus, like its activity against murine solid tumors, gemcitabine has excellent antitumor activity against a broad spectrum of human solid tumors.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/química , Citarabina/química , Citarabina/uso terapéutico , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/uso terapéutico , Humanos , Ratones , Ratones Desnudos , Modelos Biológicos , Estructura Molecular , Trasplante Heterólogo , Resultado del Tratamiento , GemcitabinaRESUMEN
Treatment of NRK-52E (normal) and H/1.2-NRK-52E (Harvey-ras transfected NRK-52E) rat kidney epithelial-like cells with two Eli Lilly antitumor compounds, sulofenur and LY295501 (15.6 microM-1000 microM) resulted in concentration- and time-dependent cell killing. Cytosolic Ca2+ became elevated in both cell lines in the presence of extracellular Ca2+ but only minimally in its absence. Both drugs were more toxic to the tumorigenic cells than to the normal cells, but LY295501 was significantly more toxic to both cells. The similarity in toxic response by both cell lines suggests a similar mechanism of toxic action for both drugs. Since LY295501 is highly toxic to tumorigenic cells but has a manageable dose-limiting toxicity it shows excellent potential for use in chemotherapy.
Asunto(s)
Antineoplásicos/toxicidad , Benzofuranos/toxicidad , Calcio/metabolismo , Riñón/metabolismo , Compuestos de Fenilurea/toxicidad , Compuestos de Sulfonilurea/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Citosol/metabolismo , Genes ras , Humanos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Neoplasias Experimentales/fisiopatología , Ratas , Células Tumorales CultivadasRESUMEN
A series of over 70 difluoropurine analogs was synthesized by varying the C-2, 6 and 8 substituents about the purine ring system. After initial in vitro and in vivo screening, testing concentrated on the 2,6-diaminopurine analog (dFdAP) and the guanosine analog (dFdG). dFDAP appears to be a prodrug for dFdG. Both compounds significantly inhibited mammary tumor growth in mice, caused a moderate inhibition in ovarian and lymphosarcoma models, and demonstrated no activity in lung and melanoma models. This is a narrower spectrum of activity than that of gemcitabine (dFdC). The antitumor activity of dFdAP in human xenografts that are refractory to standard clinical agents was comparable or superior to that of gemcitabine. However, during the preliminary toxicology testing, dFdG was associated with several deaths caused by cardiac toxicity. Therefore, although dFdG is a potentially useful oncolytic, further investigation is required.
Asunto(s)
2-Aminopurina/análogos & derivados , Antineoplásicos/farmacología , Desoxiadenosinas/química , Desoxiadenosinas/farmacología , Desoxiguanosina/análogos & derivados , Guanosina/análogos & derivados , Neoplasias Experimentales/tratamiento farmacológico , 2-Aminopurina/química , 2-Aminopurina/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Recuento de Células Sanguíneas/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Desoxiadenosinas/síntesis química , Desoxiguanosina/síntesis química , Desoxiguanosina/química , Desoxiguanosina/farmacología , Perros , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Corazón/efectos de los fármacos , Humanos , Técnicas In Vitro , Infusiones Intravenosas , Inyecciones Intraperitoneales , Hígado/efectos de los fármacos , Tejido Linfoide/efectos de los fármacos , Masculino , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Relación Estructura-Actividad , Testículo/efectos de los fármacosRESUMEN
A photoactivatable diarylsulfonylurea, N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), has been examined as a potential probe to elucidate the intracellular distribution and binding of antitumor diarylsulfonylureas. Our results demonstrated that against the human colon adenocarcinoma cell line GC3/c1, LY219703 is a more potent cytotoxic agent than N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (Sulofenur; ISCU), whereas a subline selected for resistance to ISCU was cross-resistant to LY219703, suggesting a similar mechanism of action or resistance. Cellular pharmacology studies showed that [3H]LY219703 concentrated in cells, and that its concentrative accumulation could be inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that it was similar to other antitumor diarylsulfonylurea (DSU) drugs examined. Accumulation of [3H]LY219703 in cells was progressively decreased by co-incubation with increasing concentrations of ISCU, and in cells incubated to steady state with 1 microM [3H]LY219703, ISCU (500 microM) rapidly displaced the photoaffinity analogue. Photoactivation of [3H]LY219703 by UV light (5-30 min) prevented efflux of radiolabeled drug during a 20-min wash in drug-free medium. Subsequent distribution studies showed that 89% of the radiolabel was associated with particulate components, and that approximately 20% of the radiolabel in the 320,000 g pellet could be extracted with acetone. Subcellular distribution showed approximately 6% associated with nuclei, 52% with mitochondria and 26% in the microsomal fraction. The effect of UV photoactivation on the distribution of [3H]LY219703 in soluble and particulate fractions was also examined in GC3/c1 cell preparations sonicated prior to being incubated with [3H]LY219703. A high proportion (83%) of radiolabel associated with the 100,000 g pellet, and distribution between soluble and particulate fractions was not altered by UV irradiation. Specific activities of protein in the 100,000 g supernatant and pellet were 0.186 and 0.537 nmol/mg, respectively. Putative binding species were analyzed by SDS-PAGE. Using SDS-PAGE, ten major binding proteins were identified in 320,000 g pellets from GC3/c1 cells: M(r) 110, 88, 76, 70, 64, 58, 48, 36, 26, and 24 kDa, and at least four of these (88, 70, 64, and 36 kDa) were also detected in mitochondria isolated from cells after photoactivation, or in mitochondrial preparations that were incubated with [3H]LY219703 and photoactivated after isolation from cells. Results suggested that under conditions of SDS-PAGE some dissociation of radiolabel from proteins also occurred. Binding of [3H]LY219703 to a model substrate, bovine serum albumin, and the effect of denaturing conditions used for sample preparation prior to SDS-PAGE, showed that relatively mild denaturing conditions (23 degrees, 2 hr) caused significant dissociation of radiolabel from BSA.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Adenocarcinoma/metabolismo , Antineoplásicos/metabolismo , Compuestos de Sulfonilurea/metabolismo , Marcadores de Afinidad , Sistema Libre de Células/metabolismo , Resistencia a Medicamentos , Humanos , Mitocondrias/metabolismo , Fotoquímica , Fracciones Subcelulares/metabolismo , Células Tumorales CultivadasRESUMEN
Sulofenur treatment (12.5 microM-1 mM) of colon adenocarcinoma cell lines resulted in dose- and time-dependent cell killing. LYc5 cells were viable longer than GC3/c1 cells. Each concentration resulted in elevation of cytosolic calcium [Ca2+]i) for both cell lines. At lower doses, elevation was delayed for LYc5 cells. GC3/c1 cells after 1 mM treatment in Ca(2+)-free HBSS showed no rise of [Ca2+]i. GC3/c1 cells after carbonyl cyanide-m-chlorophenylhydrazone rapidly lost rhodamine 123 fluorescence from mitochondria; after 1 mM sulofenur, fluorescence faded slowly. Following treatment, cells became rounded, blebs formed and the cells died. Results suggest that elevated [Ca2+]i plays an important role in sulofenur cytotoxicity.
Asunto(s)
Adenocarcinoma/patología , Calcio/metabolismo , Neoplasias del Colon/patología , Mitocondrias/fisiología , Compuestos de Sulfonilurea/toxicidad , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Células Tumorales CultivadasRESUMEN
Several diarylsulfonylureas (DSU), including Sulofenur (LY186641) and LY181984, have been described that exhibit wide spectrum and high therapeutic activity against murine solid tumors and human tumor xenografts. The mechanism for antitumor activity is poorly understood. Moreover, in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity for DSU. Since DSU are extensively bound to serum albumin (> 99%), we sought to determine the effect of albumin on tumor cytotoxicity. We adapted human CCRF-CEM leukemia and GC3 colon carcinoma cells for growth in UltraCHO serum- and albumin-free medium. In comparisons between normal growth medium (RPMI-1640 with 10% fetal bovine serum) and UltraCHO medium, the unbound fraction of drug correlated better with cytotoxic activity than did the total drug. Tumor cytotoxicity by DSU required > 24 h and was markedly enhanced in UltraCHO medium. For example, LY181984 and Sulofenur had IC50 values of 7.4 and 12.1 micrograms/ml against CCRF-CEM in normal growth medium and 0.6 and 0.2 microgram/ml in UltraCHO. Moreover, DSU with the lowest IC-50s in albumin-free medium displayed the most potent in vivo antitumor activity in the 6C3HED lymphosarcoma. A Sulofenur-resistant CCRF-CEM cell line was developed by culturing the cells for > 20 passages in UltraCHO medium containing LY186641 at 2 micrograms/ml (10X IC-50). This line showed approximately 18-fold resistance to LY186641, but did not show cross-resistance to vinblastine, actinomycin D, or doxorubicin. The albumin-free conditions may be useful for further mechanistic studies on the antitumor action by DSU. Further studies are underway to determine whether DSU structural requirements for cytotoxicity an albumin binding are intrinsically linked.
Asunto(s)
Antineoplásicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Albúmina Sérica/farmacología , Compuestos de Sulfonilurea/toxicidad , Animales , Línea Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Endogámicos C3H , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
We have developed a differential screening technique, single plate one transfer (SPOT), that allows the easy detection of mRNAs induced only 2-fold to 3-fold or less above background. As a model system, we looked at the induction of mRNA by parathyroid hormone (PTH) in ROS 17/2.8 rat osteosarcoma cells. The basis for this technique is to symmetrically spot in quadruplicate, on a single plate, a large number of potentially positive plaques obtained from a primary, conventional screen. We then do only one transfer from this plate in order that there will be minimal variability in DNA transfer. This filter is cut into symmetrical strips so that all clones are multiply represented on each strip. These strips are then hybridized with different probes. Since each strip contains an approximately identical amount of DNA per plaque, it is possible to accurately detect mRNAs that are induced only slightly above background. Additionally, the large sizes of the DNA plaques, as well as spotting each clone serially, contribute to the sensitivity of the technique.
Asunto(s)
Clonación Molecular/métodos , ARN Mensajero/genética , Animales , Bacteriófago lambda/genética , ADN/genética , Vectores Genéticos , Sondas Moleculares , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Ratas , Teriparatido , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Leucemia L1210/tratamiento farmacológico , Leucemia P388/tratamiento farmacológico , Leucemia Experimental/tratamiento farmacológico , Neoplasias Experimentales/tratamiento farmacológico , Células Tumorales Cultivadas/citología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Desoxicitidina/toxicidad , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Endogámicos , Células Tumorales Cultivadas/efectos de los fármacos , GemcitabinaRESUMEN
Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.
RESUMEN
The cardiotonic drugs AR-L57 [2-(2,4-dimethoxyphenyl)-1H-imidazo(4,5b)-pyridine] and isoproterenol stimulated contractility in cultured heart cells in concentration-dependent manners; only the effects of isoproterenol were blocked by propranolol. Isoproterenol, but not AR-L57, enhanced the phosphorylation state of seven protein bands [relative molecular weights (MrS) 155,000, 96,000, 27,000, 24,000, 20,000, 16,000, 12,000] and resulted in the dephosphorylation of one protein band (Mr 21,000). Also, only isoproterenol increased the activation states of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase and glycogen phosphorylase. The eight protein bands resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and detected by autoradiography were altered by isoproterenol in time- and concentration-dependent manners. The 24,000-Mr protein substrate phosphorylated in response to isoproterenol was converted to a 12,000-Mr species by heating in the presence of SDS prior to electrophoresis, suggesting that the two substrates were in fact identical proteins. A comparison of the 2-min responses to varying concentrations of isoproterenol resulted in excellent correlations between the phosphorylation states of individual protein bands and contractility. This was true even for the 21,000-Mr species that was dephosphorylated. However, only the 27,000-, 24-12,000-, and 16,000-Mr substrates were phosphorylated rapidly enough to be associated with the onset of the inotropic response. Cultured myocytes are an important feature of these studies as they are 84% pure ventricular cells that remain 100% viable throughout an experiment. Because this system is suitable for biochemical measurements and the effects of agents on heart cell contractility can be determined, it is possible to correlate changes in biochemical parameters with alterations in physiological state.
Asunto(s)
Cardiotónicos/farmacología , Imidazoles/farmacología , Isoproterenol/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida , Miosinas/metabolismo , Fosforilasas/metabolismo , Fosforilación , Propranolol/farmacología , Proteínas Quinasas/metabolismo , Estimulación QuímicaRESUMEN
It has been suggested that amrinone and AR-L57 enhance cardiac contractility either by inhibiting phosphodiesterase activity or altering Ca++ homeostasis. Because these novel agents are potentially useful in the management of heart failure, it was of interest to more clearly define their mechanism(s) of action. Amrinone and AR-L57 caused concentration-dependent increases in the contractile states of either perfused guinea-pig hearts or cultured rat cardiomyocytes. To determine whether these actions might result from an increase in sarcolemmal Ca++ movement, the effects of these agents on Ca++ accumulation were studied in a simple system, dog erythrocytes. Both agents promoted erythrocyte Ca++ accumulation in time and concentration-dependent manners, effects that resulted primarily from increased Ca++ entry. However, because these effects were not measurable at inotropic drug concentrations and were apparent only after a 30-min incubation, they did not provide an explanation for the inotropic effects of these agents. Amrinone and AR-L57 inhibited dog heart phosphodiesterase activity (isozyme III) with EC50 values of 23 and 420 microM, respectively; however, only the inotropic responses to amrinone were attenuated by the muscarinic agonist, carbachol, thereby implying a cAMP (cyclic AMP)-dependent mechanism. In cultured ventricular cells, concentrations of amrinone (2 X 10(-4) M) and AR-L57 (3 X 10(-5) M) that caused maximal inotropic responses were associated with the activation of glycogen phosphorylase, but neither drug significantly increased the activation state of cAMP-dependent protein kinase. To further probe the effects of these drugs on intracellular cAMP and Ca++ metabolism, their effects on protein phosphorylation were studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aminopiridinas/farmacología , Cardiotónicos/farmacología , Corazón/efectos de los fármacos , Imidazoles/farmacología , Amrinona , Animales , Calcio/sangre , Carbacol/farmacología , Gatos , Eritrocitos/metabolismo , Femenino , Cobayas , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocardio/enzimología , Músculos Papilares/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilasas/metabolismo , Proteínas Quinasas/metabolismo , Ratas , Ratas Endogámicas , Estimulación QuímicaRESUMEN
Chlorpromazine (CPZ) at minimally effective concentrations accumulates mammalian cells in mitosis without lethal effects on the cells. Star-metaphase morphology similar to effects seen with classical antimitotic compounds probably results from the preferential action of CPZ on a specific class of microtubules--the pole-to-pole microtubules of the mitotic spindle. At CPZ concentrations of 8 X 10(-6) M, flow cytometry indicates no effect of CPZ on the progress of cells through phases of the cell cycle other than mitosis (M). These results suggest a possible mechanism for toxic side effects of CPZ in man such as granulocytopenia and light sensitization.
Asunto(s)
Clorpromazina/farmacología , Mitosis/efectos de los fármacos , Animales , Línea Celular , Cricetinae , Cricetulus , Femenino , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Microtúbulos/ultraestructura , Ovario/citología , Ovario/efectos de los fármacosRESUMEN
Both isoproterenol and prostaglandin E1 increased the activation state of cyclic AMP-dependent protein kinase in cultured myocytes; however, only isoproterenol enhanced phosphorylase activity and contractile state. Following the incubation of intact myocytes with 32PO3-(4), 32 phosphoproteins were resolved from total cellular proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels followed by autoradiography. Isoproterenol stimulated 32PO3-(4) incorporation into 16 proteins, including 2 phosphoproteins not observed under control conditions. By contrast, prostaglandin E1 neither caused a measurable change in the protein phosphorylation pattern nor interfered with isoproterenol's capacity to do so. Isoproterenol stimulated myocyte protein phosphorylation in either the presence or absence of extracellular Ca2+. The results suggest that the regulation of protein phosphorylation following adenylate cyclase stimulation is: (1) an agonist-specific process and not due solely to a random accumulation of intracellular cycle AMP and activation of protein kinase; (2) the Ca2+ mobilization component of beta-receptor activation does not account for the paradoxical effects of isoproterenol and prostaglandin E1; (3) activation of cyclic AMP-dependent protein kinase does not always result in an enhancement of protein phosphorylation.
Asunto(s)
Isoproterenol/farmacología , Miocardio/metabolismo , Prostaglandinas E/farmacología , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Alprostadil , Animales , Animales Recién Nacidos , Células Cultivadas , AMP Cíclico/farmacología , Activación Enzimática , Ventrículos Cardíacos/metabolismo , Contracción Miocárdica , Fosforilasas/metabolismo , RatasAsunto(s)
Huesos/metabolismo , AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Animales , Huesos/ultraestructura , Calcitonina/farmacología , Células Cultivadas , Técnicas de Cultivo/métodos , Masculino , Microscopía Electrónica , Osteoblastos/ultraestructura , Hormona Paratiroidea/farmacología , Ratas , Factores de TiempoRESUMEN
Clinical experience with three vinca alkaloids currently in use as antineoplastic agents has shown a difference in the degree of peripheral neurotoxicity manifested by these compounds: vincristine greater than vindesine greater than vinblastine. This phenomenon may reflect differences in pharmacokinetics and/or the differential response of the nerve tissue itself. Differences in pharmacokinetics can be avoided by studying the direct effects of the vinca alkaloids on primary cultures of neuronal and glial cells. Vincristine at a dose as low as 0.004 microgram/ml affects the cells with processes in cultures of dissociated newborn rat midbrain. In 3-day-old cultures, after 24 h of drug treatment there is a loss of processes and swelling of the cell body. We have used this observation as the basis for a quantitative assay of the toxicity of a series of vinca compounds, and have found that for a dose range of 0.1--0.004 microgram/ml the relative toxicity of vincristine, vinblastine, and vindesine in this system correlates with their relative clinical neurotoxicity. Validation of the predictive elements of this system awaits clinical experience with novel vinca compounds.
Asunto(s)
Mesencéfalo/efectos de los fármacos , Vinblastina/análogos & derivados , Vinblastina/farmacología , Vincristina/farmacología , Animales , Células Cultivadas/efectos de los fármacos , Mesencéfalo/citología , Ratas , Factores de TiempoRESUMEN
Dissociated newborn rat superior cervical ganglion neurons in culture without exogenous nerve growth factor survive and extend processes on a monolayer of rat heart ventricular cells. An increase in the contraction rate of the heart cells was observed in 83% of the co-cultures treated with 5 X 10(-6) M tyramine. No increase was seen in heart cell cultures without neurons. These results are consistent with the assumed mode of action of tyramine--the release of catecholamines from nerve terminals--and suggest that functional interaction can occur in culture between sympathetic neurons and heart ventricular cells.