RESUMEN
NMR spectroscopy is widely used in the pharmaceutical industry for the structure elucidation of pharmaceutical impurities, especially when coupled to a separation method, such as HPLC. However, NMR has relatively poor sensitivity compared with other techniques such as mass spectrometry, limiting its applicability in impurity analyses. This limitation is addressed here through the on-line coupling of microcoil NMR with capillary isotachophoresis (cITP), a separation method that can concentrate dilute components by 2-3 orders of magnitude. With this approach, 1H NMR spectra can be acquired for microgram (nanomole) quantities of trace impurities in a complex sample matrix. cITP-NMR was used in this work to isolate and detect 4-aminophenol (PAP) in an acetaminophen sample spiked at the 0.1% level, with no interference from the parent compound. Analysis of an acetaminophen thermal degradation sample revealed resonances of several degradation products in addition to PAP, confirming the effectiveness of on-line cITP-NMR for trace analyses of pharmaceutical formulations. Subsequent LC-MS/MS analysis provided complementary information for the structure elucidation of the unknown degradation products, which were dimers formed during the degradation process.
Asunto(s)
Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Espectroscopía de Resonancia Magnética/instrumentación , Espectroscopía de Resonancia Magnética/métodos , Sistemas en Línea/instrumentación , Acetaminofén/análisis , Acetaminofén/química , Acetaminofén/aislamiento & purificación , Aminofenoles/análisis , Aminofenoles/química , Aminofenoles/aislamiento & purificación , Química Farmacéutica , Cromatografía Liquida , Estructura Molecular , Espectrometría de Masas en Tándem , Agua/químicaRESUMEN
Sample stacking techniques in electrophoresis are gaining popularity due to their ability to provide improved sensitivity and separation efficiency. The principles behind sample stacking and electrophoretic migration have been studied extensively. Nevertheless, there are still a number of observations and descriptions of ionic boundaries and migration modes for which the underlying principles are not yet fully understood. For example, the behavior of capillary isotachophoresis (cITP) systems that exhibit self-sharpening effects can be complex, especially when the buffer systems contain many ionic components. In this work, cITP coupled with 1H NMR detection is used to study electrophoretic migration of ions in both anionic and cationic cITP. A significant advantage of 1H NMR over other detection methods is the high specificity of this method, allowing detection of individual buffer and analyte constituents within the migration zones.
RESUMEN
Glycosaminoglycans (GAGs) are important in a number of biological processes and are structurally altered in many pathological conditions. The complete determination of GAG primary structures has been hampered by the lack of sensitive and specific analytical techniques. Nuclear magnetic resonance spectroscopy (NMR) is a powerful tool for GAG structure elucidation despite its relatively poor limits of detection. Solenoidal microcoils have greatly enhanced the mass limits of detection of NMR, enabling the on-line coupling of microseparation and concentration techniques such as capillary isotachophoresis (cITP), which can separate and concentrate analytes by 2-3 orders of magnitude. We have successfully used cITP coupled with on-line NMR detection to separate and concentrate nanomole quantities of heparin oligosaccharides. This sensitive on-line measurement approach has the potential to provide new insights into the relationships between biological function and GAG microstructures.
Asunto(s)
Heparina/análisis , Heparina/aislamiento & purificación , Espectroscopía de Resonancia Magnética/métodos , Oligosacáridos/análisis , Oligosacáridos/aislamiento & purificación , Sistemas en Línea , Capilares , Heparina/química , Estructura Molecular , Oligosacáridos/químicaRESUMEN
On-line capillary isotachophoresis (cITP)-NMR experiments were used to probe the interactions of the pharmaceutical compounds S-alprenolol, S-atenolol, R-propranolol, R-salbutamol and S-terbutaline with beta-cyclodextrin (beta-CD) during cITP concentration. In cITP, ionic analytes are concentrated and separated on the basis of their electrophoretic mobility. Because neutral molecules have an electrophoretic mobility of zero, they are normally not concentrated or separated in electrophoretic experiments like cITP. Most of the analytes studied were concentrated by cITP sample stacking by a factor of around 300. For analytes that formed a strong inclusion complex, beta-CD co-concentrated during cITP sample stacking. However, once the focusing process was complete, a discrete diffusional boundary formed between the cITP-focused analyte band and the leading and trailing electrolyte, which restricted diffusion into and out of the analyte band.
Asunto(s)
Ciclodextrinas/análisis , Electroforesis/métodos , Espectroscopía de Resonancia Magnética/métodos , Albuterol/análisis , Albuterol/química , Alprenolol/análisis , Alprenolol/química , Atenolol/análisis , Atenolol/química , Ciclodextrinas/química , Difusión , Electrólitos/química , Propranolol/análisis , Propranolol/química , Protones , Sensibilidad y Especificidad , Terbutalina/análisis , Terbutalina/químicaRESUMEN
OBJECTIVE: To evaluate the safety of IDN-6556, a novel anti-apoptotic pan-caspase inhibitor, administered in single and multiple ascending doses in normal volunteers and patients with hepatic dysfunction. MATERIALS AND METHODS: IDN-6556 was administered as a 30-minute intravenous infusion in rising doses to 3 groups: Group A, normal volunteers, given as a single infusion, Group B, normal volunteers, given q.i.d. for 7 days, Group C, patients with hepatic impairment (elevated transaminases, alanine transaminase, ALT and aspartate transaminase, AST), given q.i.d. for 7 days. RESULTS: The drug was well tolerated up to 10 mg/kg/infusion for a single dose, and 1.5 mg/kg/infusion q.i.d. for 7 days, with the dose-limiting adverse event of phlebitis or inflammation at the site of the infusion. This toxicity was predicted from animal studies. Clinically and statistically meaningful dose-related falls in transaminases were seen in all but 1 of the hepatic impaired patients. Two-way ANOVA analyses of the changes for all the IDN-6556 groups combined versus placebo were: ALT absolute change: p < 0.0001 and % change: p = 0.012, AST absolute and % changes: p < 0.0001. After discontinuation of the drug (after 7 days of dosing), the transaminases rapidly returned to the pre-treatment levels. CONCLUSIONS: Following intravenous administration of a novel anti-apoptotic caspase inhibitor, adverse events were mild-to-moderate in severity, resolved in a few days and did not result in any subject terminating treatment prematurely. The effects in hepatic impaired patients appear to be consistent with both the administration and subsequent abrupt withdrawal of an effective hepatoprotective drug that delays cell death in hepatocytes.
Asunto(s)
Inhibidores de Caspasas , Inhibidores Enzimáticos/uso terapéutico , Hepatopatías/tratamiento farmacológico , Hígado/efectos de los fármacos , Adulto , Apoptosis/efectos de los fármacos , Área Bajo la Curva , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/farmacocinética , Femenino , Semivida , Humanos , Hígado/enzimología , Hepatopatías/enzimología , Masculino , Transaminasas/efectos de los fármacos , Transaminasas/metabolismoRESUMEN
OBJECTIVES: To evaluate whether caspases are involved in ethanol (EtOH)-induced apoptosis and if polyenylphosphatidylcholine (PPC) affects apoptosis, in vitro in Hep G2 cells. METHODS: Cells were treated with 100 mmol/L EtOH for 24 h and with 2 doses of 100 mmol/L EtOH (1/24 h) in the presence of absence of 20 mmol/L of PPC or 50 micromol/L caspase 3 inhibitor (IDN). Cells were analyzed for apoptosis by transmission electron microscopy (TEM) 6000 cells/treatment, DNA fragmentation by ELISA and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (T dt-mediated d-UTP) nick-end-labeling, TUNEL. RESULTS: 100 mmol/L dose of EtOH resulted in 22 +/- 2.5% (p < 0.001) apoptosis (vs. control). Two consecutive doses of 100 mmol/L EtOH for 24 h each caused 36 +/- 3.0% (p < 0.001 vs. control and p < 0.05 vs. one dose). PPC significantly reduced apoptosis (vs. non exposed to PPC): 100 mmol/L -12 +/- 1.5% (p < 0.05) and 2 x 10(-)(0) mmol/L -20 +/- 2.0% (p < 0.001). Pretreatment with 50 micromol caspase inhibitor reduced EtOH-induced apoptosis in a similar proportion. CONCLUSIONS: PPC downregulates EtOH-apoptosis by a mechanism similar to caspase inhibition.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Reparación del ADN/efectos de los fármacos , Etanol/farmacología , Transducción de Señal , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Microscopía ElectrónicaRESUMEN
Protection of ischemic myocardium is an important unmet need in reperfusion therapy of acute myocardial infarction. Myocardial ischemia and reperfusion induce necrosis and apoptosis in cardiomyocytes. Caspase processing and activation are critical steps in most receptor and nonreceptor pathways of apoptosis. Caspase inhibitors have been shown to reduce ischemia reperfusion injury in cardiac muscle. Information about dose response and time of administration are needed to optimize the design of preclinical studies. We used isolated adult rabbit cardiomyocytes subjected to metabolic inhibition (MI) and recovery to examine the role of caspases and caspase inhibitors, the dose response, and the timing of administration. In vitro inhibitory concentrations (Ki) were determined for purified caspases. Cardiomyocytes subjected to MI were treated with peptidomimetic fluoromethyl ketone inhibitors of caspases before or during MI, or at recovery. Caspase inhibitors were most effective when added before MI and included throughout recovery, but were partially protective if added after MI. The optimal concentration of the inhibitors tested was approximately 10 microM. Protection was sustained when cells were allowed to recover for 4 or 24 h. These results suggest that caspase activation is an important component of myocyte injury mediated by MI and recovery. Low doses of caspase inhibitors were identified that reduce injury in this model system, and further investigations using in vivo models are warranted.
Asunto(s)
Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Corazón/efectos de los fármacos , Miocardio/enzimología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Miocardio/citología , Conejos , Transducción de SeñalRESUMEN
Previous studies have shown that caspase inhibitors are effective at protecting against anti-Fas antibody (alpha-Fas)-mediated liver injury/lethality. The purpose of these experiments was to characterize more fully the efficacy of a broad-spectrum, irreversible caspase inhibitor, IDN-1965 (N-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid), in this model and the role of caspase inhibition in long-term protection. The ED(50) for IDN-1965 by i.p. administration, based on alanine aminotransferase activities, was 0.14 mg/kg. The caspase inhibitor was also efficacious when administered intravenously and orally (ED(50) values of 0.04 and 1.2 mg/kg, respectively). Histologically, marked reduction in Fas-induced apoptosis with IDN-1965 (1 mg/kg, i.p.) was apparent at 6 h. Also, caspase 3-like activities were decreased in a dose-dependent manner, but the inhibition of caspase activity was transient. Immunohistochemical studies demonstrated that IDN-1965 greatly reduced the activation of caspase 3. In survival studies, a single i.p. treatment of 1 mg/kg IDN-1965 or continuous i.p. infusion via osmotic pumps completely blocked lethality measured up to 7 days after alpha-Fas administration. IDN-1965 was also effective in inhibiting liver injury when administered as long as 3 h after or 1 h before alpha-Fas administration. Lastly, Western blot analysis demonstrated that processing of caspases 3, 6, and 8, as well as Bid (a protein responsible for the release of mitochondrial cytochrome C and amplification of the apoptotic cascade) was inhibited by IDN-1965. In conclusion, the broad-spectrum caspase inhibitor IDN-1965 is markedly effective at inhibiting Fas-mediated apoptosis by multiple routes of administration. The therapeutic potential of caspase inhibitors appears promising for the treatment of apoptosis-mediated liver injury based on potency and postinsult efficacy.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Inhibidores de Cisteína Proteinasa/farmacología , Indoles/farmacología , Oligopéptidos/farmacología , Alanina Transaminasa/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Receptor fas/genéticaRESUMEN
The typical proliferative response of hepatocytes to tumor necrosis factor (TNF) can be converted to a cytotoxic one by transcriptional arrest. Although NF-kappaB activation is critical for hepatocyte resistance to TNF toxicity, the contribution of other TNF-inducible transcription factors remains unknown. To determine the function of c-Myc in hepatocyte sensitivity to TNF, stable transfectants of the rat hepatocyte cell line RALA255-10G containing sense and antisense c-myc expression vectors were isolated with increased (S-Myc cells) and decreased (AN-Myc cells) c-Myc transcriptional activity. While S-Myc cells proliferated in response to TNF treatment, AN-Myc cells underwent 32% cell death within 6 h. Fluorescent microscopic studies indicated that TNF induced apoptosis and necrosis in AN-Myc cells. Cell death was associated with DNA hypoploidy and poly(ADP-ribose) polymerase cleavage but occurred in the absence of detectable caspase-3, -7, or -8 activation. TNF-induced, AN-Myc cell death was dependent on Fas-associated protein with death domain and partially blocked by caspase inhibitors. AN-Myc cells had decreased levels of NF-kappaB transcriptional activity, but S-Myc cells maintained resistance to TNF despite NF-kappaB inactivation, suggesting that c-Myc and NF-kappaB independently mediate TNF resistance. Thus, in the absence of sufficient c-Myc expression, hepatocytes are sensitized to TNF-induced apoptosis and necrosis. These findings demonstrate that hepatocyte resistance to TNF is regulated by multiple transcriptional activators.
Asunto(s)
Apoptosis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Caspasas/metabolismo , Línea Celular , Activación Enzimática , Hepatocitos/citología , Hepatocitos/patología , FN-kappa B/metabolismo , Necrosis , Oligonucleótidos Antisentido/genética , Proteínas Proto-Oncogénicas c-myc/genética , RatasRESUMEN
Reactive oxygen intermediates (ROI) have been implicated as mediators of hepatocyte death resulting from a variety of forms of liver injury. To delineate the mechanisms that underlie ROI-induced apoptosis, the roles of caspase activation and nuclear factor-kappaB (NF-kappaB) signaling were determined in the rat hepatocyte cell line RALA255-10G after treatment with H(2)O(2) or the superoxide generator menadione. By 8 h, H(2)O(2) and menadione caused 26% and 33% cell death, respectively. Death from both ROI occurred by apoptosis as indicated by morphology under fluorescence microscopy, the induction of caspase activation and DNA fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Despite the presence of caspase activation in both forms of apoptosis, caspase inhibition blocked H(2)O(2)- but not menadione-induced apoptosis. In contrast, inhibition of NF-kappaB activation decreased cell death from both ROI. Different ROI, therefore, induce distinct apoptotic pathways in RALA hepatocytes that are both caspase dependent and independent. In contrast to the known protective effect of NF-kappaB activation in tumor necrosis factor-alpha-induced hepatocyte apoptosis, NF-kappaB promotes hepatocellular death from ROI in these cells.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Peróxido de Hidrógeno/farmacología , Hígado/citología , Hígado/fisiología , FN-kappa B/metabolismo , Superóxidos/farmacología , Vitamina K/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 2 , Caspasa 3 , Caspasa 7 , Caspasa 8 , Caspasa 9 , Muerte Celular/efectos de los fármacos , Línea Celular , Hígado/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Caspasas/metabolismo , Hígado/fisiología , Factor de Necrosis Tumoral alfa/toxicidad , Animales , Proteínas Portadoras/metabolismo , Inhibidores de Caspasas , Línea Celular , Grupo Citocromo c/metabolismo , Dactinomicina/farmacología , Activación Enzimática , Proteína de Dominio de Muerte Asociada a Fas , Proteínas I-kappa B , Hígado/citología , FN-kappa B/metabolismo , Ploidias , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Transducción de SeñalRESUMEN
Axon guidance mechanisms are crucial to the development of an integrated nervous system. One family of molecules that may be important in establishing axonal connectivity in mammals is the Netrins, and their putative receptors DCC (deleted in colorectal cancer), Neogenin, and Unc-5. Knockout and mutational analyses of some of these genes have shown that they are critically involved in the development of several specific pathways in the developing brain. However, previous expression analyses of these genes have largely been confined to the developing spinal cord. In the present study, we analyzed the expression of DCC in the developing mouse forebrain. We found that DCC protein is expressed in specific axonal populations projecting from the developing olfactory bulb, neocortex, hippocampus, and epithalamus/habenular complex. In the developing olfactory bulb and neocortex, DCC expression is particularly evident during the targeting phase of axon outgrowth and is then rapidly downregulated. As predicted from the knockout and mutational analyses of this gene, DCC is expressed in axonal commissures, in particular the corpus callosum, hippocampal commissure, and the anterior commissure. In addition, we found that DCC is expressed in the habenular commissure, the fasciculus retroflexus, and the stria medularis. Therefore, this analysis implicates a function for DCC in additional axonal guidance systems not predicted from the knockout and mutational analyses.
Asunto(s)
Axones/metabolismo , Moléculas de Adhesión Celular/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Prosencéfalo/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor , Animales , Receptor DCC , Femenino , Masculino , Ratones , Receptores de Netrina , Netrina-1 , Embarazo , Prosencéfalo/embriologíaRESUMEN
Ceramide has been implicated as a second messenger in intracellular signaling pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 to 100 micromol/L C2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-kappaB (NF-kappaB) activation. Because ceramide may mediate cell death from tumor necrosis factor alpha (TNF-alpha), the ability of RNA synthesis inhibition and NF-kappaB inactivation to sensitize hepatocytes to ceramide toxicity was examined. RALA hepatocytes were sensitized to ceramide toxicity by coadministration of actinomycin D (ActD). Cell death occurred by apoptosis as determined by the presence of morphological evidence of apoptosis, caspase activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypoploidy. Despite the induction of apoptosis associated with caspase activation, cell death from ActD/ceramide was not blocked by caspase inhibition. Inhibition of NF-kappaB activation also sensitized RALA hepatocytes to ceramide toxicity, but to a lesser extent than for TNF-alpha. Thus, unlike many nonhepatic cell types, rat hepatocytes are resistant to cell death from ceramide because of the transcriptionally dependent up-regulation of a protective gene(s). The ability of ActD and NF-kappaB inactivation to sensitize RALA hepatocytes to ceramide toxicity suggests that ceramide may act as a downstream mediator of TNF-alpha toxicity.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Dactinomicina/farmacología , Hígado/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Esfingosina/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores Enzimáticos/toxicidad , Indoles/farmacología , Hígado/patología , Hígado/fisiología , Masculino , FN-kappa B/metabolismo , Oligopéptidos/farmacología , Ploidias , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Ratas Sprague-Dawley , Esfingosina/antagonistas & inhibidores , Esfingosina/toxicidadRESUMEN
BACKGROUND: Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC. METHODS: Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr. RESULTS: Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor. CONCLUSION: During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.
Asunto(s)
Caspasas/farmacología , Endotelio/citología , Hígado , Hígado/citología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Precursores Enzimáticos/metabolismo , Indoles/farmacología , Hígado/efectos de los fármacos , Hígado/lesiones , Trasplante de Hígado , Oligopéptidos/farmacología , Preservación de Órganos , Ratas , Daño por Reperfusión/enzimología , Daño por Reperfusión/etiologíaRESUMEN
Calcium influx is believed to play a critical role in the cascade of biochemical events leading to neuronal cell death in a variety of pathological settings, including cerebral ischemia. The synthetic omega-conotoxin peptide SNX-111, which selectively blocks depolarization-induced calcium fluxes through neuronal N-type voltage-sensitive calcium channels, protected the pyramidal neurons in the CA1 subfield of the hippocampus from damage caused by transient forebrain ischemia in the rat model of four-vessel occlusion. SNX-111 provided neuroprotection when a single bolus injection was administered intravenously up to 24 hr after the ischemic insult. These results suggest that the window of opportunity for therapeutic intervention after cerebral ischemia may be much longer than previously thought and point to the potential use of omega-conopeptides and their derivatives in the prevention or reduction of neuronal damage resulting from ischemic episodes due to cardiac arrest, head trauma, or stroke. Microdialysis studies showed that SNX-111 was 3 orders of magnitude less potent in blocking potassium-induced glutamate release in the hippocampus than the conopeptide SNX-230, which, in contrast to SNX-111, failed to show any efficacy in the four-vessel occlusion model of ischemia. These results imply that the ability of a conopeptide to block excitatory amino acid release does not correlate with its neuroprotective efficacy.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Maleato de Dizocilpina/farmacología , Hipocampo/efectos de los fármacos , Ataque Isquémico Transitorio/fisiopatología , Neuronas/efectos de los fármacos , Péptidos/farmacología , Prosencéfalo/efectos de los fármacos , omega-Conotoxinas , Animales , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Glutamatos/metabolismo , Ácido Glutámico , Hipocampo/metabolismo , Hipocampo/patología , Ataque Isquémico Transitorio/patología , Masculino , Neuronas/patología , Péptidos/síntesis química , Potasio/farmacología , Prosencéfalo/patología , Tractos Piramidales/efectos de los fármacos , Tractos Piramidales/metabolismo , Tractos Piramidales/patología , Ratas , Ratas Endogámicas F344 , Reperfusión , Factores de TiempoRESUMEN
We examined the effects of omega-conopeptides, a novel class of neuronal voltage-gated calcium channel antagonists, on hemodynamic responses in rats. Intravenous (i.v.) injections of SNX-111 (omega-conopeptide MVIIA) dose-dependently decreased arterial blood pressure (BP) in conscious rats. Intracerebroventricular (i.c.v.) SNX-111 injections (580 pmol) tended to increase BP and, after an initial decrease, to increase heart rate (HR). The dose-response curve for SNX-111 administered i.v. in conscious rats was biphasic. Beginning at subdepressor doses, SNX-111 caused a long-lasting blockade of pressor responses elicited by sympathetic nerve stimulation in pithed animals but did not prevent increases in BP evoked by exogenously administered norepinephrine (NE). Pretreatment of rats with histamine antagonists partially blocked the hypotensive effects of the higher (870 and 2,900 nmol/kg) doses of SNX-111. Substitution of alanine for arginine at position 10 ([Ala10]-MVIIA) markedly attenuated the histamine-mediated component of the vasodepressor response. Together, these findings indicate that SNX-111 administered i.v. decreases systemic BP by a combination of blockade of sympathetic neurotransmission and mast cell degranulation; the latter function appears to be dependent on the arginine residue in position 10 of the amino acid sequence of SNX-111.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Péptidos Cíclicos/farmacología , Péptidos/farmacología , omega-Conotoxinas , Secuencia de Aminoácidos , Animales , Clorfeniramina/farmacología , Cimetidina/farmacología , Estado de Descerebración , Relación Dosis-Respuesta a Droga , Inyecciones Intraventriculares , Masculino , Datos de Secuencia Molecular , Norepinefrina/farmacología , Péptidos/administración & dosificación , Péptidos Cíclicos/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
The possible occurrence of NPK-LI in the ventral horns of the embryonic chicken spinal cord was investigated by means of the indirect immunofluorescence method. The results showed a transient appearance of NPK-LI in cells of the lateral motor column between day 5 of incubation and hatching. After this they disappeared and in the ventral horns NPK-LI remained only in fibers. The results are discussed in terms of a possible trophic action of NPK during development.
Asunto(s)
Embrión de Mamíferos/metabolismo , Embrión no Mamífero , Neuropéptidos/metabolismo , Médula Espinal/embriología , Taquicininas , Animales , Embrión de Pollo , Embrión de Mamíferos/citología , Desarrollo Embrionario y Fetal , Técnica del Anticuerpo Fluorescente , Neuronas Motoras/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
A polyclonal antiserum raised against the insulin-like growth factor-II (IGF-II) receptor has been used to map the distribution of this receptor in the developing rat central nervous system (CNS). Transiently high levels of receptor immunoreactivity were found in the developing brain, particularly in the cortex and hypothalamus. The amount of receptor immunostaining in these areas decreases toward the time of birth, and levels are approximately equivalent to those in the adult by postnatal day 7. The choroid plexus, cerebral vasculature, ependymal cells, retina, and pituitary contain high levels of receptor immunoreactivity throughout embryogenesis and adulthood. Some mesodermally derived tissues, such as bone, also demonstrate transient expression of IGF-II receptor during fetal development. These data are consistent with potential roles for IGF-II in CNS development, in the development of specific mesodermal tissues, and in specific regions of the postnatal CNS.
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Encéfalo/crecimiento & desarrollo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Receptores de Superficie Celular/metabolismo , Somatomedinas/metabolismo , Médula Espinal/crecimiento & desarrollo , Animales , Encéfalo/irrigación sanguínea , Encéfalo/embriología , Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Plexo Coroideo/metabolismo , Epéndimo/metabolismo , Hipotálamo/metabolismo , Técnicas para Inmunoenzimas , Mesodermo/metabolismo , Hipófisis/metabolismo , Ratas , Ratas Endogámicas , Receptores de Somatomedina , Retina/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismo , Distribución TisularRESUMEN
Nerve fibres displaying such immunoreactivity were revealed by indirect immunofluorescence. Neuropeptide K-like immunoreactive fibres, entering the pulp within large nerve trunks, were distributed around blood vessels as well as in the stroma. Some immunoreactive fibres were also observed in the para-odontoblastic region. In view of the biological activity of neuropeptide K, it is tentatively proposed that it may act in the dental pulp as a regulatory peptide involved in neurogenic inflammation, blood flow regulation and sensory transmission.
Asunto(s)
Pulpa Dental/análisis , Neuropéptidos/análisis , Taquicininas , Vasos Sanguíneos/inervación , Pulpa Dental/irrigación sanguínea , Pulpa Dental/inervación , Humanos , Inmunohistoquímica , Fibras Nerviosas/análisis , Fibras Nerviosas/ultraestructuraRESUMEN
The functional role of striatonigral neurokinins were studied by analysing the effects of intranigral injections of substance P and neurokinin A on the extracellular level of dopamine and dihydroxyphenylacetic acid in the striatum, as measured by in vivo microdialysis in rats. Two substance P antagonists, substance P D-Pro2 D-Trp7,9 and substance P D-Arg1 D-Trp7,9 Leu11 were tested and analysed for their ability to block the neurokinin effects. Unilateral injections of substance P (0.00007-7.0 nmol injected in 0.2 microliter) as well as neurokinin A (0.009-9.0 nmol) into the substantia nigra, pars reticulata of halothane anaesthetized rats produced long-lasting increases in ipsilateral striatal dopamine and dihydroxyphenylacetic acid levels. The dose-response relationship for substance P on dopamine was biphasic, with maximal effects occurring after the middle dose (0.007-0.07 nmol). The dose-response relationship for neurokinin A was monophasic. Intranigral injections of substance P D-Pro2 D-Trp7,9 (0.07-0.7 nmol) or substance P D-Arg1 D-Trp7,9 Leu11 (0.07-0.7 nmol) produced a decrease in striatal dopamine, but an increase in striatal dihydroxyphenylacetic acid. At a low dose (0.07 nmol) substance P D-Pro2 D-Trp7,9 enhanced the dopamine increase produced by intranigral substance P (0.07 nmol) or neurokinin A (0.09), while at a high dose (0.7 nmol) it blocked both substance P and neurokinin A effects. Both doses of substance P D-Arg1 D-Trp7,9 Leu11 (0.07 and 0.7 nmol) blocked the substance P- but not the neurokinin A-induced increase in striatal dopamine. Immunohistochemical analysis revealed that high doses of substance P (7.0 nmol) and neurokinin A (0.9 and 9.0 nmol), as well as substance P D-Pro2 D-Trp7,9 and substance P D-Arg1 D-Trp7,9 Leu11 (0.07 and 0.7 nmol), induced a restricted loss of tyrosine hydroxylase in dendrites and cells, and neuropeptide K in terminals, at the site of injection. Further analysis shows that co-administration of substance P (0.07 nmol) or neurokinin A (0.09 nmol) did not modify the extent of the depletion of both immunoreactivities induced by substance P D-Arg1 D-Trp7,9 Leu11 (0.7 nmol). The extent of the effect produced by substance P D-Arg1 D-Trp7,9 Leu11 (0.7 nmol) was, however, smaller than the spread of intranigral injection of [125I]Bolton-Hunter-labelled substance P D-Arg1 D-Trp7,9 Leu11, and it is suggested that the "neurotoxic" effects of the substance P antagonists are not primarily involved in their abilities to inhibit striatal dopamine release and block the stimulation of dopamine after intranigral substance P and neurokinin A.(ABSTRACT TRUNCATED AT 400 WORDS)