[Renin release from kidney slices, as a function of the electrolyte composition of the medium].
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Animals↗
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[Mechanism of liver cell damage due to alkylphosphate].
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Adenosine Diphosphate↗
Inhibition by oxotremorine of acetylcholine resting release from guinea pig-ileum longitudinal muscle strips.
1. Longitudinal muscle strips of the guinea-pig ileum were incubated in Tyrode solution containing either DFP or physostigmine as cholinesterase inhibitor. After a 90 min preincubation period the acetylcholine resting release into the medium was determined. Acetylcholine was estimated by gas chromatography. 2. The resting release was 0.39 nmol/g times min irrespective of the cholinesterase inhibitor used. In the presence of hexamethonium, or after omission of external calcium, the resting release fell by 50 and 55 per cent, respectively. 3. Oxotremorine (10-5 and 10-4M) significantly inhibited the resting release of acetylcholine by 25 and 33 per cent, respectively. The inhibitory effect of oxotremorine was completely reversed by atropine (3 times 10-7 M). Oxotremorine did not reduce the spontaneous release of acetylcholine that occurred either in the presence of hexamethonium or in the absence of external calcium. 4. The acetylcholine content of the muscle strips was approximately doubled during the preincubation with a cholinesterase inhibitor. The subsequent incubation with oxotremorine did not lead to a further increase in the endogenous acetylcholine content. However, incubation of the muscle strips with oxotremorine in the absence of a cholinesterase inhibitor led to a rise in the endogenous acetycholine concentration. In in vivo experiments, oxotremorine also caused an increase in the acetylcholine content of the muscle strips. The possibility is discussed that the rise in the acetylcholine concentration following the administration of oxotremorine is a consequence of the decreased release. 5. It is concluded that oxotremorine inhibits the resting release of acetylcholine by activation of neuronal muscarinic receptors. The inhibitory effect of exotremorine is linked to that fraction of the acetylcholine resting release that is calcium-dependent and that arises from propagated activity in cholinergic neurones. The results are consistent with the hypothesis of a feed-back control of acetylcholine release mediated by inhibitory muscarinic receptors.
Acetylcholine↗
Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: developmental profiles, in vivo and in cell culture, and recovery after inactivation.
1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.
Acetylcholinesterase↗
Failure of guinea pig plasma kallikrein to cause neutrophil migration.
Guinea pig plasma kallikrein was apparently chemotactic for guinea pig neutrophils when assayed in Boyden's chamber. However, it was concluded that this phenomenon was artificial due to the following reasons: Kallikrein was only chemotactic in the presence of bovine serum albumin (BSA) in the chamber but not when BSA was substituted by guinea pig serum albumin or egg albumin. Kallikrein injection failed to cause dermal tissue leukocytosis. Kallikrein did not polarize neutrophils. A chemoattractant(s) seemed to be generated from BSA preparation time dependently. Although this generation required the enzymatic activity of kallikrein, the chemotactic activity once generated was resistant to a kallikrein inhibitor (diisopropylfluorophosphate) and antiprekallikrein antibody.
Animals↗
Production of macrophage activation factors by tryptic cleavage of calf serum proteins.
Trypsin, when added to a bioassay for tumoricidal macrophages, produced killing of tumor cells. Trypsin cleaved fetal calf serum proteins to produce a protein fragment that activated macrophages to lyse tumor cells. Diisopropyl fluorophosphate-inhibited trypsin did not produce tumoricidal macrophages either by direct action on the macrophage or by action on serum proteins. The macrophage activation factors produced from serum proteins were fractionated into molecular weight ranges of 150,000, 68,000, and 30,000-5000. The effects of neutral proteinases and proteinase inhibitors on the ability of macrophages to lyse tumor cells is discussed.
Animals↗
Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases.
The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1-0.5 microM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophosphate (DPF; 2.5 mM)--although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 microM) additively enhanced the dithiothreitol (DTT; 1.0 mM)--increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 microM) enhanced the effect of Ca2+ (10 microM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 microM) was significantly decreased by the presence of calpastatin (24 micrograms/ml), an inhibitor of Ca(2+)-activated neutral protease (calpain). Now, regucalcin (0.25 microM) increased about 7-fold the activity of m-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.
Animals↗
In vivo and in vitro effects of diisopropyl fluorophosphate and paraoxon on individual molecular forms of rat brain acetylcholinesterase.
Previous study in this laboratory showed that following a sc injection of an organophosphorus compound, diisopropyl fluorophosphate (DFP), into rats the inhibition of 10S molecular forms was considerably more pronounced than that of 4S forms of brain acetylcholinesterase (AChE). This could depend on different accessibility of the two forms or on their different intrinsic sensitivity to the antiChE compound. In the present study the effects of DFP and Paraoxon on 10S and 4S forms were evaluated in vivo, i.e., after systemic administration, and in vitro by adding the organophosphorus compounds to each of the two forms after extraction from brain of untreated rats, solubilization and separation. The in vivo preferential inhibition of 10S forms was confirmed. The 10S/4S ratios for control and DFP-treated rats were 9.05 and 5.01, respectively; these ratios were 8.46 and 3.33 for Paraoxon. On the other hand, in the in vitro experiments there were no significant differences between IC50 values for 10S and 4S forms both in the case of DFP (2.66 and 2.98 microM) and Paraoxon (32.4 and 42.4 nM, respectively). The overall data suggest that the preferential in vivo inhibition of 10S molecular forms with respect to 4S forms depends on their different accessibility probably due to different subcellular localization of the two forms and not on their different intrinsic sensitivity.
Acetylcholinesterase↗
In vivo and in vitro effects of diisopropyl phosphorofluoridate (DFP) on the rate of hen brain tubulin polymerization.
Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in sensitive species. We have investigated the in vivo and in vitro effects of DFP on hen brain tubulin polymerization. Hens were treated with a single dose of DFP (1.7 mg/kg, sc.), and were sacrificed after 18-21 days. Tubulin from DFP-treated hen brains showed small but significant decrease (14.42%) in the rate of polymerization and 11.05% decrease in rise in O.D. at 340 nm in 30 min. DFP in vivo treatment also resulted in decreased concentration of tau and an enhanced concentration of two peptides (45 kDa, 35 kDa) in the brain supernatant. These peptides seemed to be the degradation products of MAP-2. The decrease in the rate of brain tubulin polymerization in treated hens is consistent with neurochemical alterations and the focal degeneration and aggregation of these filamentous structures in OPIDN.
Animals↗
Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism.
Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats, confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.
Abnormalities, Drug-Induced↗
Comparison of Ca2+/calmodulin-dependent protein kinase II purified from control and diisopropyl phosphorofluoridate (DFP)-treated hens.
Diisopropyl phosphorofluoridate (DFP) produces type I organophosphorus ester-induced delayed neurotoxicity in humans and sensitive animal species. This is accompanied by enhanced Ca2+/CaM-dependent protein kinase II (CaM-kinase II) activity, and [125I]calmodulin binding to CaM-kinase II in DFP-treated hen brain supernatant without increase in the enzyme quantity. We have purified CaM-kinase II from control and DFP-treated hen whole brains and compared various physical and biochemical properties. The two enzymes exhibited similar properties in many respects. However, there was a decrease in calcium-independent protein kinase II activity after autophosphorylation, and an increase in K0.5 for free calcium and calmodulin of enzyme purified from DFP-treated hen brains. This change in kinetic parameters may result in greater percentage of total CaM-kinase II present in unphosphorylated form, which is consistent with the increased autophosphorylation of CaM-kinase II and [125I]calmodulin binding in the brain supernatant of DFP-treated hens.
Animals↗
Preferential inhibition of acetylcholinesterase molecular forms in rat brain.
The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G4), and monomeric (G1) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G1 than for the G4 form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G1 form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.
Acetylcholinesterase↗
Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment.
The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P2) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P1, P2, and P3) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000 g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.
Acetylcholinesterase↗
Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase.
A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammonium-chloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight approximately 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme. A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.
Acetylcholinesterase↗
Neurofilament phosphorylation and [125I]calmodulin binding by Ca2+/calmodulin-dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)-treated hen.
Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animal species, e.g., adult chicken. The chickens were sacrificed 18 days after a single dose of DFP (1.7 mg/kg, s.c.), which produced severe ataxia or paralysis in 10-14 days. We studied Ca2+/calmodulin-dependent in vitro neurofilament phosphorylation by the brain subcellular fractions of control and DFP-treated hens. There was enhanced phosphorylation of all three NF subunits by the brain supernatant of treated hens. This was accompanied by enhanced autophosphorylation of both Ca2+/CaM-dependent protein kinase II (CaM-kinase II) subunits and increased calmodulin binding using either 125I-CaM or biotinylated calmodulin to only alpha subunit without concomitant increase in the amount of this enzyme. This enhanced phosphorylation of neurofilament subunits was completely and partially inhibited by mastoparan and KN-62, respectively. There was no alteration in the distribution of CaM-kinase II activity in treated hens and the activity was not related to its concentration in different subcellular fractions. The difference in 125I-CaM binding to CaM-kinase II alpha subunit in the brain supernatants of control and DFP-treated hens was not altered by its phosphorylation or dephosphorylation. The increased CaM-kinase II activity in the soluble fraction of DFP-treated hen brain may be involved in the aberrant phosphorylation of axonal neurofilaments, and thus play a role in OPIDN.
Animals↗
Effect of neutral proteases from human granulocytes on colony forming cells in vitro.
Marked inhibition of colony formation is observed after incubation of mouse and human bone marrow cells with the human granulocytic neutral proteases elastase and chymotrypsin as well as with pancreatic chymotrypsin. The corresponding enzymes inactivated with diisopropylfluorophosphate were almost inactive. Incubation of different colony inducing agents either resulted in no change or in an increase of their colony stimulating activity. The data suggest a direct proteolytic action of the proteases on colony forming cells which may alter receptor sites for colony stimulating activities.
Animals↗
Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors.
The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2-4 months) and aged (22-24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [3H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [3H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.
Aging↗
Hormone-sensitive lipase in brown adipose tissue: identification and effect of cold exposure.
Hormone-sensitive lipase (HSL) in brown adipose tissue from mice was identified through immunoprecipitation with a polyclonal antibody (anti-HSL) towards rat white fat HSL and Western blotting. An 82 kDa polypeptide, slightly smaller than the rat white fat HSL 84 kDa subunit, was detected and its identity as HSL verified by inhibition properties. The HSL concentration per g tissue was several-fold higher in the mouse brown adipose tissue than in the rat white adipose tissue, but the specific activities per mg protein were similar. Cold-exposure (4 degrees C) of the mice for 24 h approximately doubled the HSL concentration but this increase parallelled the overall protein increase and did not reflect a specific effect on the HSL.
Adipose Tissue, Brown↗