RESUMEN
The ability of bromine and rat liver microsomes (RLM) to convert organophosphorus (OP) protoxicants to esterase inhibitors was determined by measuring acetylcholinesterase (AChE) and neuropathy target esterase (NTE) inhibition. Species specific differences in susceptibility to esterase inhibition were determined by comparing the extent of esterase inhibition observed in human neuroblastoma cells and hen, bovine, and rodent brain homogenates. OP protoxicants examined included tri-o-tolyl phosphate (TOTP), O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), leptophos, fenitrothion, fenthion, and malathion. Bromine activation resulted in greater AChE inhibition than that produced by RLM activation for equivalent concentrations of fenitrothion, malathion, and EPN. For EPN and leptophos, bromine activation resulted in greater inhibition of NTE than RLM. Only preincubation with RLM activated TOTP; resultant inhibition of AChE was less in hen brain (13 +/- 3%) than in neuroblastoma cells (73 +/- 1%) at 10(-6) M. In contrast, 10(-6) M RLM-activated TOTP produced more inhibition of hen brain NTE (89 +/- 6%) than NTE of human neuroblastoma cells (72 +/- 7%). Human neuroblastoma cells and brain homogenates from hens, the accepted animal model for study of OP-induced neurotoxicity, were relatively similar in sensitivity to esterase inhibition. Homogenates from hens were more sensitive to NTE inhibition induced by phenyl saligenin phosphate (PSP), an active congener of TOTP, than were homogenates from less susceptible species (mouse, rat, bovine). AChE of hen brain homogenates was also more sensitive than homogenates from other species to malaoxon, the active form of malathion.
Asunto(s)
Encéfalo/metabolismo , Inhibidores de la Colinesterasa/farmacocinética , Inhibidores de la Colinesterasa/toxicidad , Insecticidas/farmacocinética , Insecticidas/toxicidad , Neuroblastoma/metabolismo , Compuestos Organofosforados/farmacocinética , Compuestos Organofosforados/toxicidad , Compuestos Organotiofosforados , Profármacos/farmacocinética , Profármacos/toxicidad , Animales , Biotransformación , Bromo/metabolismo , Bromo/farmacología , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Bovinos , Pollos , Humanos , RatasRESUMEN
In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests.
Asunto(s)
Inhibidores Enzimáticos/toxicidad , Esterasas/antagonistas & inhibidores , Microsomas Hepáticos/efectos de los fármacos , Compuestos Organofosforados/toxicidad , Animales , Bromo/toxicidad , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Cloropirifos/toxicidad , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/toxicidad , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/metabolismo , Humanos , Masculino , Neuroblastoma , Compuestos Organofosforados/metabolismo , Paratión/toxicidad , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.
Asunto(s)
Acetilcolinesterasa/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Neoplasias Encefálicas/enzimología , Hidrolasas de Éster Carboxílico/efectos de los fármacos , Hidrolasas de Éster Carboxílico/metabolismo , Neuroblastoma/enzimología , Compuestos Organofosforados/farmacología , Animales , Relación Dosis-Respuesta a Droga , Esterasas/antagonistas & inhibidores , Humanos , Ratones , Neuronas/efectos de los fármacos , Neuronas/enzimología , Compuestos Organofosforados/toxicidad , Células Tumorales CultivadasRESUMEN
The differential inhibition of the target esterases acetylcholinesterase (AChE) and neuropathy target esterase (NTE, neurotoxic esterase) by organophosphorus compounds (OPs) is followed by distinct neurological consequences in exposed subjects. The present study demonstrates that neuroblastoma cell lines (human SH-SY5Y and murine NB41A3) can be used to differentiate between neuropathic OPs (i.e., those inhibiting NTE and causing organophosphorus-induced delayed neuropathy) and acutely neurotoxic OPs (i.e., those highly capable of inhibiting AChE). In these experiments, concentration-response data indicated that the capability to inhibit AChE was over 100x greater than the capability to inhibit NTE for acutely toxic, nonneuropathic OPs (e.g., paraoxon and malaoxon) in both cell lines. Inhibition of AChE was greater than inhibition of NTE, without overlap of the concentration-response curves, for OPs which are more likely to cause acute, rather than delayed, neurotoxic effects in vivo (e.g., chlorpyrifos-oxon, dichlorvos, and trichlorfon). In contrast, concentrations inhibiting AChE and NTE overlapped for neuropathy-causing OPs. For example, apparent IC50 values for NTE inhibition were less than 9.6-fold the apparent IC50 values for AChE inhibition when cells were exposed to the neuropathy-inducing OPs diisopropyl phosphorofluoridate, cyclic tolyl saligenin phosphate, phenyl saligenin phosphate, mipafox, dibutyl dichlorovinyl phosphate, and di-octyl-dichlorovinyl phosphate. In all cases, esterase inhibition occurred at lower concentrations than those needed for cytoxicity. These results suggest that either mouse or human neuroblastoma cell lines can be considered useful in vitro models to distinguish esterase-inhibiting OP neurotoxicants.
Asunto(s)
Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Inhibidores de la Colinesterasa/toxicidad , Insecticidas/toxicidad , Sistema Nervioso/efectos de los fármacos , Neuroblastoma/patología , Compuestos Organofosforados , Animales , Biomarcadores , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Sistema Nervioso/enzimología , Neuroblastoma/enzimología , Especificidad de la Especie , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Two knit glove fabrics, one of 100% cotton and one of 100% polypropylene, were examined for their capability to decrease the penetration of the organophosphate insecticides (OPs), azinphos-methyl and paraoxon after 4 h at field concentrations (3000 and 15 ppm, respectively) through an in vitro epidermal system (Skin2, Advanced Tissue Systems, LaJolla, CA). The glove fabrics were examined under three different conditions of use: new, after they had been abraded and after they had been abraded and then laundered. New and laundered cotton fabric was also examined for its capability to decrease the penetration of azinphos-methyl through another in vitro epidermal system (Epiderm, MatTek Corp., Ashland, MA), after 4 and 24 h of exposure. Capability of the media under the in vitro epidermal systems to inhibit brain acetylcholinesterase (AChE) was used as the indicator of penetration. Results were compared to OP-caused inhibitions seen in media under the fabric alone and in media under the in vitro epidermal systems alone. Incubations of azinphos-methyl suspensions and the in vitro epidermal systems covered with fabric indicated that both the epidermal cells and fabric provided protection against AChE inhibition caused by this OP and that the protective effects were additive, whether measured after 4 or 24 h of exposure. Therefore, neither laundering nor abrasion followed by laundering altered the capability of the in vitro epidermal systems to absorb azinphos-methyl suspension. For paraoxon solution, however, new cotton glove fabric prevented absorption, and this protective effect, noted after 4 h of exposure, was lost when the fabric was laundered. Abrading the fabric did not cause a greater effect than laundering alone. These results suggest that the pesticide as well as its formulation may be factors of consideration when protective fabrics are chosen, and that, for cotton glove fabric, the protection against some OPs may best be provided before the fabric is laundered.
Asunto(s)
Azinfosmetilo/metabolismo , Guantes Protectores/normas , Insecticidas/metabolismo , Paraoxon/metabolismo , Absorción , Acetilcolinesterasa/análisis , Inhibidores de la Colinesterasa/metabolismo , Gossypium/metabolismo , Técnicas In Vitro , Lavandería/normas , Polipropilenos/metabolismo , Polipropilenos/normas , Piel ArtificialRESUMEN
Blood samples and vascular segments from the ischiadic artery of hens treated with either cyclic phenyl saligenin phosphate (PSP; 2.5 micrograms/kg, im) or paraoxon (PXN; 0.1 micrograms/kg, im) in the presence or absence of verapamil, a calcium channel antagonist (7 micrograms/kg, im, given 4 consecutive days beginning the day before PSP or PXN administration), were examined 1, 3, 7, and 21 d after PSP or PXN administration in order to determine the contribution of catecholamines and peripheral blood vessel physiology and morphology to organophosphorus-induced delayed neuropathy (OPIDN). The levels of plasma catecholamines were measured by high-performance liquid chromatograpy (HPLC) and indicated a different effect with PSP, which causes OPIDN, and PXN, which does not. PSP treatment elevated the levels of norepinephrine and epinephrine throughout the study, while PXN treatment depressed the levels of these catecholamines. Verapamil treatment attenuated the OP response by approximately 50% for both compounds. Ischiadic vessel segments were isolated from OP-treated hens and perfused at a constant flow rate of 12 ml/min, then examined for their response to potassium chloride (KCl, 3 x 10(-3) M), acetylcholine (ACh), phenylephrine (PE), an alpha 1 adrenergic agonist, and salbutamol (SAL), a beta 2 adrenergic agonist. Agents were delivered in concentrations of 10(-8) to 10(-3) M. Vascular segments did not respond to ACh or SAL at any concentration used. Vessels displayed a significant reduction in contractile response to both KCl (3 x 10(-3) M) and PE (10(-8) to 10(-3) M) 3 and 21 d after exposure to either PSP or PXN. This reduced response was not altered by the presence of verapamil. Innervation of the peripheral vasculature was unchanged after OP treatment. This study indicates that plasma catecholamine levels could be differentially altered by treatment with OPs that do and do not cause OPIDN and suggests that the alterations involve intracellular calcium. In contrast, vascular response of the ischiadic artery was altered following OP treatment, but the effect was not specific for the neuropathy-inducing OP, PSP, and response was not mediated by Ca 2+, nor was it the result of autonomic nerve deterioration.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Epinefrina/sangre , Insecticidas/toxicidad , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/sangre , Verapamilo/farmacología , Acetilcolina/farmacología , Acetilcolinesterasa/sangre , Antagonistas Adrenérgicos beta/farmacología , Albuterol/farmacología , Animales , Arterias , Alcoholes Bencílicos/administración & dosificación , Alcoholes Bencílicos/toxicidad , Biomarcadores/sangre , Bloqueadores de los Canales de Calcio/administración & dosificación , Hidrolasas de Éster Carboxílico/sangre , Pollos , Relación Dosis-Respuesta a Droga , Femenino , Insecticidas/administración & dosificación , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Paraoxon/administración & dosificación , Paraoxon/toxicidad , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , Distribución Aleatoria , Verapamilo/administración & dosificaciónRESUMEN
Certain organophosphorus compounds (OPs) produce a delayed neuropathy (OPIDN) in man and some animal species. Capability to cause OPIDN is generally predicted in animal models by early and irreversible inhibition of neuropathy target esterase (NTE, neurotoxic esterase). In this study, NTE inhibition in response to OP exposure was examined in cell culture, using the human SH-SY5Y neuroblastoma cell line. Cells were exposed for 1 hr to equimolar (1 x 10(-5) M) concentrations of 6 OPs associated with OPIDN in vivo (including 2 protoxicants and 4 active (-P = O) toxicants), and 8 OPs that do not produce delayed neuropathy in animal models (including 5 protoxicants and 3 -P = O compounds). The -P = O compounds that cause OPIDN in animal models inhibited NTE > 60% at the test concentration; -P = O compounds that do not cause OPIDN in animal models inhibited NTE < 30%. Protoxicants did not inhibit NTE at the test concentration, reflecting their limited metabolism in the human cell line. These results indicate that human neuroblastoma cells have potential use in the initial screening of bioactive OPs with capability for causing OPIDN.
Asunto(s)
Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Neuroblastoma/enzimología , Compuestos Organofosforados/toxicidad , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Ésteres , Humanos , Enfermedades del Sistema Nervioso Periférico/enzimología , Células Tumorales CultivadasRESUMEN
Cotton and synthetic knit glove fabrics in combination with an in vitro skin model were used to examine the capability of fabric to decrease the dermal absorption of the organophosphorus insecticides azinphos-methyl, paraoxon, and malathion. Capability for inhibition of acetylcholinesterase was determined in samples of media taken from under the skin barrier after the skin model, with or without fabric protection, had been exposed to the test compounds for 4 h. Acetylcholinesterase inhibitions caused by the direct addition of organophosphorus insecticide to the media were also included in the comparison. Results indicated that the skin model system alone had some capability to serve as a barrier to the transfer of organophosphates. Fabric covering used on the test model increased the barrier between insecticide application and resultant acetylcholinesterase inhibition. The all-cotton, 7-cut knit was especially effective in preventing the absorption of azinphos-methyl, as this organophosphorus insecticide had no capability to cause acetylcholinesterase inhibition when this fabric was used to protect the skin model. Knit glove materials of 100% cotton were demonstrated to be effective in preventing the absorption of paraoxon and malathion. These studies indicate that an in vitro model system can be used in combination with fabrics to study the relationship between clothing and skin as barriers to the absorption of organophosphorus insecticides.
Asunto(s)
Guantes Protectores , Insecticidas/toxicidad , Compuestos Organofosforados , Absorción Cutánea , Textiles , Gossypium , Humanos , Técnicas In Vitro , Nylons , PoliésteresRESUMEN
The pharmacokinetics of pentoxifylline (P) and its alcohol metabolite I (MI) were determined after administration of intravenous pentoxifylline, sustained release pentoxifylline tablets (Trental), and crushed pentoxifylline tablets in corn syrup, to five healthy adult horses. Pharmacokinetics were evaluated in a model-independent manner. After intravenous administration, pentoxifylline was rapidly eliminated (mean residence time 1.09 +/- 0.67 h), had a large steady-state volume of distribution (2.81 +/- 1.16 l/kg), and high clearance (3.06 +/- 1.05 l/kg/h). Oral absorption of pentoxifylline from both dose forms varied considerably between individuals. Times to peak concentration ranged from 1-10 h for either dose form. There was no difference in relative bioavailability (F') between whole (0.98 +/- 0.30) and crushed Trental tablets. Ratios between areas under the curve (AUC) for pentoxifylline and MI were different following administration of oral versus intravenous doses. This finding suggests that route of administration may affect the metabolic profile of pentoxifylline. Given the extreme differences in absorption characteristics between individuals in this study, recommendations are not made as to appropriate dose, dose interval, or dose form for administration of pentoxifylline to horses.
Asunto(s)
Caballos/metabolismo , Pentoxifilina/farmacocinética , Absorción , Administración Oral , Animales , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión/veterinaria , Preparaciones de Acción Retardada , Vías de Administración de Medicamentos , Inyecciones Intravenosas , Pentoxifilina/administración & dosificación , ComprimidosRESUMEN
Carbaryl, a carbamate insecticide, exerts its toxic effect in animals by inhibiting the activity of neural acetylcholinesterase. Differences in sensitivity of this enzyme to inhibition were studied after intraperitoneal administration to chickens and rats. A dose of 900 mg/kg to chickens and 70 mg/kg to rats caused equivalent inhibition of brain cholinesterase activities (57% +/- 6 and 47% +/- 4, respectively) 60 min after administration, which was the time of maximal cholinergic signs. Signs of toxicity (salivation, respiratory distress, muscle tremors and weakness) were more pronounced in rats than in chickens when brain acetylcholinesterase was inhibited to the same extent in both species. Carboxylesterase activities in brain, liver, and plasma were also inhibited 60 min after administration of carbaryl to chickens and rats. Activities of enzymes associated with hepatic microsomes were unaffected. Specific activities of brain esterases, including acetylcholinesterase, carboxylesterase and neurotoxic esterase, were higher in untreated chickens than in untreated rats. Specific activities of liver esterases (carboxylesterase, A-esterase) were, however, 4- and 10-fold lower in untreated chickens than in untreated rats. Total clearance of carbaryl in the chicken, determined after intravenous administration of 5 mg/kg, was 0.26 +/- 0.02 l/kg/min. This value is 5.7 times higher than that reported for the rat, indicating that the relatively lower activities of esterases in the liver of chickens did not affect the clearance of this chemical in the avian species.
Asunto(s)
Carbaril/farmacocinética , Animales , Carbaril/toxicidad , Pollos , Inhibidores de la Colinesterasa/farmacocinética , Inhibidores de la Colinesterasa/toxicidad , Relación Dosis-Respuesta a Droga , Esterasas/antagonistas & inhibidores , Femenino , Masculino , Ratas , Ratas Endogámicas , Especificidad de la EspecieRESUMEN
A microtiter plate reader with an associated computer to average triplicate samples and subtract blanks was used for reading and calculating neurotoxic esterase (NTE, also known as neuropathy target esterase) activities in spinal cord regions of hens 4 hr after administration of diisopropylphosphorofluoridate (DFP, 0.5 mg/kg sc). Although NTE inhibition is an early indicator of organophosphorus ester-induced delayed neuropathy. DFP-induced inhibition was not greater in regions of the spinal cord where pathological changes are most notable. Acetylcholinesterase (AChE) activities and protein determinations were also done on these tissues using microassay methods. DFP-induced AChE inhibition was similar to NTE inhibition. In addition to the capability to be used for small regional esterase activity measurements, the microassay was advantageous because the number of samples incorporated into a single assay was increased and the time needed for the NTE assay was reduced by 50%. Total volume of incubate in each well was 0.3 ml; the incubate contained 1/20 quantities of sample and reagents necessary in more conventional assays. Validation of the microassay was performed by comparison with more conventional assays when measuring inhibition of NTE and AChE in brains of control and experimental hens of two different genetic strains (B13B13 and B21B21 white leghorns). Experimental birds were given DFP, 0.5 mg/kg sc, 24 hr before samples were collected. NTE activities in brains of control hens were similar using both types of NTE analytical procedures. Percentage inhibition of NTE caused by DFP was within 4% using both assay procedures in both strains of hens.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hidrolasas de Éster Carboxílico/análisis , Acetilcolinesterasa/análisis , Animales , Encéfalo/enzimología , Pollos , Femenino , Indicadores y Reactivos , Isoflurofato , Microquímica , Ratas , Nervio Ciático/enzimología , Médula Espinal/enzimologíaRESUMEN
The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN/efectos de la radiación , Metanosulfonato de Etilo/farmacología , Metilnitrosourea/farmacología , Mutación , Rayos Ultravioleta , Animales , Cafeína/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cricetinae , Cricetulus , ADN/efectos de los fármacos , Femenino , Hipoxantina Fosforribosiltransferasa/genética , Cinética , OvarioRESUMEN
Activity of calcium-activated neutral protease (CANP or calpain), an enzyme responsible for degradation of axonal and muscle cytoskeletal elements, was determined in brain, sciatic nerve, and gastrocnemius muscle of hens given tri-ortho-tolyl phosphate (TOTP, 360 mg/kg po) or active congener phenyl saligenin phosphate (PSP, 2.5 mg/kg im) with and without a calcium channel blocker which ameliorated clinical signs of organophosphate-induced delayed neuropathy (nifedipine 1 mg/kg/day x 5). Calcium channel blocker administration was initiated 1 day prior to administration of organophosphate (OP). OP administration caused an increase in CANP activity in brain within 4 days and in sciatic nerve and gastrocnemius muscle within 2 days of administration. This increase did not occur if nifedipine was administered to PSP-treated hens. Total sciatic nerve calcium concentrations were also increased by PSP, but not until OP-treated hens were no longer being administered calcium blockers. This indicates that calcium channel blockers may contribute to amelioration of organophosphate-induced delayed neuropathy by attenuation of calcium-mediated disruption of axonal and muscle cytoskeletal homeostasis.
Asunto(s)
Encéfalo/enzimología , Calpaína/análisis , Cresoles/toxicidad , Músculos/enzimología , Nifedipino/farmacología , Nervios Periféricos/enzimología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Tritolilfosfatos/toxicidad , Animales , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Pollos , Femenino , Enfermedades del Sistema Nervioso Periférico/enzimologíaRESUMEN
A mutant form of the type I regulatory subunit (RI) of cAMP-dependent protein kinase has been cloned and sequenced (Clegg, C. H., Correll, L. A., Cadd, G. C., and McKnight, G. S. (1987) J. Biol. Chem. 262, 13111-13119) which contains two point mutations in the site B cAMP-binding site, a Gly to Asp at position this report, the effect of each independent mutation on the rate of dissociation of cAMP from RI, the cAMP-mediated activation of holoenzyme and the inducibility of cAMP-responsive genes has been characterized. Dissociation of cAMP from either recombinant wild type RI or the B1 mutant demonstrated biphasic kinetics, indicating two sites with different affinities for cAMP. Dissociation from the B2 subunit, however, was monophasic and very rapid indicating that site B had been destroyed and that the rate of dissociation from site A was increased. The cAMP activation constants (Ka) of the wild type and B1 holoenzymes were 40 and 188 nM, respectively, and demonstrated positive cooperativity, with Hill coefficients of 1.61 for the wild type and 1.67 for B1. The B2 holoenzyme required much greater concentrations of cAMP, 4.7 microM, for half-maximal activation and did not display positive cooperativity. Constitutive expression in mouse AtT20 pituitary cells of the B1 mutant resulted in only a small shift in the Ka for kinase activation in these cells compared with B2 expression which increased the Ka by more than 100-fold. Transient expression of the B1 subunit in human JEG-3 choriocarcinoma cells inhibited forskolin activation of a cAMP-responsive promoter by 35% whereas similar expression of the B2 RI subunit inhibited the response by 90%. These results suggest that the Gly to Asp mutation at amino acid 324 completely blocks cAMP binding to site B whereas the Arg to His mutation at position 332 causes a more subtle alteration in cAMP binding. Expression of either mutant RI in animal cells results in a dominant repression of cAMP-dependent protein kinase activity and cAMP-dependent protein kinase-mediated processes.
Asunto(s)
AMP Cíclico/metabolismo , Mutación , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , ADN/genética , Activación Enzimática , Genes , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Plásmidos , Unión Proteica , Proteínas Quinasas/genética , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.
Asunto(s)
Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intracelular , Mutación , Proteínas Quinasas/genética , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , AMP Cíclico/fisiología , Activación Enzimática , Escherichia coli/genética , Vectores Genéticos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
cAMP-dependent protein kinase (PKA; ATP: protein phosphotransferase; EC 2.7.1.37) appears to be the major mediator of cAMP responses in mammalian cells. We have investigated the role of PKA subunits in the regulation of specific genes in response to cAMP by cotransfection of wild-type or mutant subunits of PKA together with cAMP-inducible reporter genes. Overexpression of catalytic subunit induced expression from three cAMP-regulated promoters (alpha-subunit, c-fos, E1A) in the absence of elevated levels of cAMP but did not affect expression from two unregulated promoters (Rous sarcoma virus, simian virus 40). Cotransfection of a regulatory subunit gene containing mutations in both cAMP binding sites strongly repressed both basal and induced expression from the cAMP-responsive alpha-subunit promoter without affecting expression from the Rous sarcoma virus promoter. These experiments indicate that cAMP induces gene expression through phosphorylation by the catalytic subunit and that the ambient degree of phosphorylation dictates the level of basal as well as induced expression of the cAMP-regulated alpha-subunit gene.
Asunto(s)
Regulación de la Expresión Génica , Genes , Proteínas Quinasas/metabolismo , Transcripción Genética , Animales , Virus del Sarcoma Aviar/genética , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Colforsina/farmacología , Globinas/genética , Cinética , Luciferasas/genética , Sustancias Macromoleculares , Ratones , Plásmidos , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , TransfecciónRESUMEN
The effect of the microsomal enzyme inducer beta-naphthoflavone (beta NF) on the development of organophosphorus-induced delayed neuropathy (OPIDN) was examined in two laboratories (VPI and MSU), utilizing two strains of White Leghorn hens. A single intraperitoneal injection of beta NF at 80 mg/kg body weight 48 h prior to administration of o-tolyl saligenin phosphate (TSP), the neuroactive metabolite of tri-o-tolyl phosphate (TOTP), caused a significant increase in hepatic microsomal cytochrome P-450 concentrations and aniline hydroxylase activities after 72 h in both strains. Hepatic carboxylesterase and cholinesterase activities were not affected by beta NF treatment in either strain. Administration of TSP in single subcutaneous doses of 20 and 25 mg/kg body weight (VPI) or 30 and 60 mg/kg body weight (MSU) caused significant inhibition of whole-brain neuropathy target esterase (NTE) activity 24 h postdosing, and hens subsequently developed clinical signs characteristics of OPIDN. beta NF had no significant effect on NTE inhibition or on initiation or severity of OPIDN clinical signs. However, OPIDN clinical signs were less severe in the strain of bird (MSU) with the higher intrinsic hepatic carboxylesterase activity and the higher beta NF-induced cytochrome P-450 concentration. The study indicates that microsomal enzyme induction, which has been shown to alleviate TOTP-induced delayed neuropathy, could not alleviate OPIDN resulting from exposure to TSP. This study also suggests that strain may affect susceptibility to TSP-induced delayed neuropathy.